CN119143861B - Mannan-binding lectin-altering protein ppg-rep-MBL, DNA molecule, expression vector, host cell and application - Google Patents
Mannan-binding lectin-altering protein ppg-rep-MBL, DNA molecule, expression vector, host cell and application Download PDFInfo
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Abstract
本发明涉及一种甘露聚糖结合凝集素改造蛋白ppg‑rep‑MBL、DNA分子、表达载体、宿主细胞，以及用途，属于生物医药技术领域。本发明提供了一种甘露聚糖结合凝集素改造蛋白ppg‑rep‑MBL，其氨基酸序列如 SEQ ID NO:3 所示，核苷酸序列如 SEQ ID NO:4 所示。本发明还提供了该甘露聚糖结合凝集素改造蛋白ppg‑rep‑MBL在制备预防或/和治疗冠状病毒感染的药物中的用途。本发明改造蛋白不与MASP2蛋白结合，从而明显减弱对补体凝集素途径的活化作用，同时保留其与甘露糖的结合特性，可以有效结合并阻断新型冠状病毒假病毒及其Delta突变株对HEK293T‑ACE2 细胞的侵染，为该蛋白被设计为开发广谱抗冠状病毒感染的抑制剂或药物提供候选可能。
The present invention relates to a mannan-binding lectin modified protein ppg-rep-MBL, a DNA molecule, an expression vector, a host cell, and uses, and belongs to the field of biomedical technology. The present invention provides a mannan-binding lectin modified protein ppg-rep-MBL, whose amino acid sequence is shown in SEQ ID NO:3 and whose nucleotide sequence is shown in SEQ ID NO:4. The present invention also provides the use of the mannan-binding lectin modified protein ppg-rep-MBL in the preparation of a drug for preventing or/and treating coronavirus infection. The modified protein of the present invention does not bind to the MASP2 protein, thereby significantly weakening the activation effect on the complement lectin pathway, while retaining its binding properties with mannose, and can effectively bind to and block the infection of the new coronavirus pseudovirus and its Delta mutant strain to HEK293T-ACE2 cells, providing a candidate possibility for the protein to be designed as an inhibitor or drug for the development of a broad-spectrum anti-coronavirus infection.
Description
Technical Field
The invention relates to a mannan-binding lectin-modified protein ppg-rep-MBL, a DNA molecule, an expression vector, a host cell and application thereof in preparing medicines for preventing or/and treating coronavirus infection, belonging to the technical field of biological medicines.
Background
Mannan-binding lectin (MBL), an important component of innate immunity, an intrinsic component in the complement activation process (complement lectin pathway), has attracted considerable attention for its recognition and regulatory function in viral infections. The human MBL has obvious antibacterial and antiviral activities and good drug-forming property. From the rationality of developing new drugs, MBL as a drug to be applied to the human body itself will have the advantage of low immunogenicity as compared to foreign proteins. However, if MBL is injected into a large amount of human body in the form of a pharmaceutically acceptable protein, the problems of overactivation of complement lectin pathway (complement system) and excessive promotion of phagocytosis of phagocytes are necessarily caused, various adverse reactions are caused, and the human body is caused to generate inflammatory injury and even death. Thus, balancing or controlling complement system activation and inhibition of adverse reactions during severe disease processes with inflammation, but without disrupting its protective function, is becoming an important issue in current research.
Current research reports on MBL focus mainly on the function of specific sequences. As reported by Ying L et al, three point mutations in the collagen-like region (CLR) of the human MBL gene CGT 52 TGT, GGC 54 GAC, and GGA 57 GAA resulted in a significantly lower binding capacity of MBL mutant proteins to mannans than wild-type MBL proteins, and MBL mutants were not able to efficiently activate complement lectin pathway .（Ying L ,Feng-Ling L ,Zhi-Jun B , et al.Defective activities, but not secretions, resulting from gene point mutations of human mannan-binding lectin.[J].Molecular medicine reports,2012,5(4): 1121-7.）Russell W et al, two mutations were made to rat MBL (G25D and G28E). The G28E mutant was found to activate complement more than 10-fold less efficiently than the G25D mutant, while the G25D mutant was 7-fold less active than the wild-type MBL. It was further found that the reduced complement activation by both mutant MBLs was due to the failure of MASP-2 to be effectively activated when bound to the mannan-coated surface. Kurata H et al purified and identified human MBL in both liver MBL (L-MBL) and serum MBL (S-MBL) form from liver and serum, respectively, and found that newly synthesized MBL in human liver was processed into the above two forms after translation, differing by 9 amino acids. And synthesizing recombinant human MBL in COS-1 cells, which was found to lack a sequence comprising 9 amino acid residues (Ser-Ser-Pro-Gly-Ile-Asn-Gly-Phe-Pro) at the front end of the collagen-like domain, while the protein was not capable of activating complement, suggesting that this sequence plays an important role in the human MBL complement activation process .（Kurata H, Sannoh T, Kozutsumi Y, Yokota Y, Kawasaki T. Structure and function of mannan-binding proteins isolated from human liver and serum. J Biochem. 1994 Jun;115(6):1148-54.）Girija, U. V et al found the lysine residue at the X position of the Gly-X-Y collagen repeat sequence, i.e., lys (56) in ficolin-A, which is critical for MASP-2 binding, was found in all ficolin and MBL known to activate complement, while a recombinant protein was prepared with double mutations of K56P and M57O, data indicating that Lys56 and/or Met57 are critical for binding to MASP-2, is critical for effective MASP activation. In rat MBL, the equivalent mutant (K46P and V47O) MBL proteins also eliminate MASP-2 binding and complement activation .（Girija, U. V., Dodds, A. W., Roscher, S., Reid, K. B.,&Wallis, R. (2007). Localization and characterization of the mannose-binding lectin (MBL)-associated-serine protease-2 binding site in rat ficolin-A: equivalent binding sites within the collagenous domains of MBLs and ficolins. Journal of immunology (Baltimore, Md. : 1950), 179(1), 455–462.）Daming Zuo et al designed and synthesized a series of peptides based on the CLR sequence in human MBL that were thought to block MBL-MASP interactions to localize serine protease binding motifs on human MBL. The results indicate that MASP binds to the C-terminus located in the hinge region of the collagen-like domain (formed by disruption of Gly-X-Y repeats). in addition, R32C, the G35D and G37E mutant proteins have similar serine protease binding properties to wild type MBL, but the mutant MBL protein binds to MASPs much weaker than the wild type MBL protein binds to MASPs, application number CN201910560238.2, entitled recombinant protein comprising human IgG1Fc and mannan-binding lectin C-terminus, recombinant protein comprising human IgG1Fc and mannan-binding lectin C-terminus and polynucleotide encoding the protein are provided. The invention also provides a preparation method for expressing the recombinant protein. The concentration of the recombinant protein expressed by plasmid transfected cells containing the polynucleotide with the optimized sequence is 1.50mg/ml after purification, which is 3 times of the concentration of the expressed protein of the coding sequence with the non-optimized codon. The recombinant protein has good binding activity with 5 candida standard strains and clinical strains which account for more than 90 percent of fungal infection in blood, and the recombinant protein and 5 candida can be combined, and the combination is mediated by CRD region of the recombinant protein MBL. Chen Ade et al studied the effect of mannan-binding lectin (MBL) on human neutrophil (PMN) function. MBL has been shown to enhance PMN phagocytosis in a complement lectin pathway dependent manner and promote inflammatory cytokine secretion (mannan-binding lectin effects on human neutrophil function J South Med Univ, 2013, 33 (6): 842-846).
MBL is a multimeric mannose/mannoglycan sugar binding protein, the single chain of which is divided into 4 parts of an N-terminal, a collagen-like region (CLR), a neck region and a sugar recognition region (CRD), the N-terminal is the key of polymerization of 3 monomers of the MBL protein, the neck and CRD regions are the key of whether the MBL can recognize and bind to pathogens, the amino acid sequence of the MBL is SEQ ID NO.1, and the nucleotide sequence of the MBL is SEQ ID NO. 2. During complement lectin pathway activation, the premise of complement activation is that the recognition and binding of the OGKXGP short motif in MBL-CLR by mannan-binding lectin associated serine protease 2 (MASP 2) （Ichise,Steven H. Sacks, Development of a collagen-like peptide polymer via end-to-end disulfide cross-linking and its application as a biomaterial[J]. Acta Biomaterialia, 2019, 9(4): 361-371）,, if substituted or deleted, results in the failure of CLR to form the collagen-like subunit structure, thereby affecting other biological functions of MBL proteins.
There is no report on the weakening/eliminating of MBL activating complement lectin pathway, and there is no report on the weakening/eliminating of MBL activating complement lectin pathway and the retention of mannose/mannose binding property.
Infection by the novel coronavirus (SARS-CoV-2) may induce excessive activation of the immune system and release of high concentrations of pro-inflammatory cytokines, resulting in "cytokine storms". Such excessive production of inflammatory cytokines may lead to disease progression, organ failure, acute respiratory distress syndrome, and death. In this context, it is also important to study how to cope with viral infections, and how to develop drugs using the line of immune defense and its related proteins, for the human immune system, in particular the innate immune response and its complement system.
Currently, there are patents reporting that MBL gene is applied to SARS-CoV detection reagent (patent number: CN 200510082997.0) and recombinant MBL is applied to new coronal detection kit (patent application number: CN 202311287315.4), recombinant MBL is used to block new coronavirus infection (patent number: CN 202110490146.9) and MBL polypeptide, or functional fragment, derivative, mutant thereof is used to inhibit new coronavirus infection (patent number: US20240123029A 1).
There is no report on the use of low immunogenic MBL engineered proteins that reduce/prevent activation of the complement system for the prevention or/and treatment of coronavirus infections.
Disclosure of Invention
The present invention provides a modified protein ppg-rep-MBL with low immunogenicity capable of inhibiting coronavirus infection, which is an MBL modified protein which is successfully expressed by the inventor through modifying the gene of MBL, reduces complement system activation and simultaneously ensures the sugar binding capacity of the MBL modified protein. The invention also provides a preparation method and application of ppg-rep-MBL.
The invention provides a mannan-binding lectin-modifying protein ppg-rep-MBL, the amino acid sequence of which is shown as SEQ ID NO. 3, and the nucleotide sequence of which is shown as SEQ ID NO. 4.
The engineered proteins are prepared by replacing the OGKXGP short motif in the collagen-like region (CLR region) of MBL that recognizes and binds to MASP2, thereby activating the complement pathway, by reference to the biofunctional sequence CCCPP [ GPP ]4GPR [ GPP ]4GCCC of native collagen and by retaining the motif sequence GEKGEP that mediates binding to C1qR, thereby enhancing phagocytosis of phagocytes in vivo. The invention introduces the collagen sequence to replace MBL-CLR region to ensure the formation of the original glue, but in order to make the length of the replaced MBL protein amino acid sequence more approximate to that of the natural MBL protein amino acid sequence, the invention repeats the PP [ GPP ]4GPR [ GPP ]4G sequence when introducing the collagen sequence, namely, CCCPP [ GPP ]4GPR [ GPP ]4G sequence, only the N end 3 cysteines are reserved, the C end 3 cysteines are removed, thus not only leading the N end Cys to crosslink the triple helix through disulfide bond, but also not changing the bond angle of the C end CRD region specificity recognition sugar residue of the MBL. And to ensure the functional integrity of the engineered MBL protein, the in situ motif sequence GEKGEP is maintained at the C-terminus of the sequence. It was finally replaced with CCCPP GPP 4GPR GPP 4GGEKGEP (ppg-rep peptide fragment).
The invention also provides application of the mannan-binding lectin-modifying protein ppg-rep-MBL in preparing medicines for preventing or/and treating coronavirus infection.
Wherein the drug is a drug blocking SRAS-CoV-2 infection.
Wherein the SARS-CoV-2 comprises an original strain and a mutant strain.
The invention also provides a preparation method of the mannan-binding lectin-modifying protein ppg-rep-MBL, which comprises the following steps:
a. Constructing recombinant manna binding lectin ppg-rep-MBL amino acid sequence by using wild manna binding lectin gene sequence SEQ ID NO. 1 as template and nucleic acid sequence SEQ ID NO. 2, replacing the MBL-CLR region of amino acid sequence of wild manna binding lectin with CCCPP GPP 4GPR 4GGEKGEP, designing and synthesizing ppg-rep-MBL gene, the amino acid sequence is SEQ ID NO. 3 and the nucleotide sequence is SEQ ID NO. 4;
b. Construction of an engineered mannan-binding lectin recombinant plasmid the ppg-rep-MBL gene was inserted between HindIII and XbaI restriction sites of vector pCMV3 to construct plasmid pCMV3-ppg.
C. the mannan-binding lectin recombinant protein ppg-rep-MBL was prepared.
The ratio between cells, plasmid and transfection reagent was calculated according to the transfection reagent instructions, DMEM and plasmid were mixed in one sterile EP tube, DMEM and PEI transfection reagent were mixed in the other sterile EP tube and left at room temperature for 5min. Mixing the liquids in the two EP pipes, turning over, mixing, standing at room temperature for 20min, adding a proper amount of the mixed liquid into 293T cells to be transfected, and culturing the cells in a cell culture incubator at 37 ℃ for 6-8h under the condition of 5% CO 2. After 6h, 1% ITS, 60. Mu.M/L VC was added. After further culturing for 72 hours, cell supernatants containing ppg-rep-MBL protein were harvested and PMSF was added to one thousandth of the supernatant volume. And (3) packaging the supernatant by using a dialysis bag (MD 44) with the aperture of 8-12 kDa, uniformly spreading PEG20000, concentrating, and taking out until the volume is about 1/10 of the original volume. The 500 mu lMannan-Agarose protein purification column is placed in a10 ml column filter, and the column is sequentially rinsed with 15-20 times of ultrapure water and 15 times of TBS-Ca 2+ buffer. After draining the buffer, the column was resuspended with 2 column volumes of TBS-Ca 2+ buffer, the ppg-rep-MBL protein sample was mixed with Mannan-Agarose purification column in a ratio of 100 μl column per 1ml sample, and the mixture was placed on a vertical mixer and hung on the column overnight at 4 ℃. The next day, put into a10 ml column filter, drain the redundant liquid, wash the column with TBS-Ca 2+ buffer, and wash the impurity protein. The ppg-rep-MBL protein was eluted with 20mmol/LEDTA of TBS-Ca 2+ and collected in a centrifuge tube. Ultrafiltration is performed by using an ultrafiltration tube with a pore diameter of 30kDa, centrifugation is performed at 4000g for 10min at 4 ℃ and liquid inside the ultrafiltration tube is retained. Dialyzing with 20mmolTris-HCl and 150mmolNaCl (pH=7.4) dialysate overnight, and changing dialysate every 6-8 hr to obtain target protein.
The invention also provides a DNA molecule which codes for the mannan-binding lectin-altering protein ppg-rep-MBL.
The invention also provides an expression vector containing the DNA molecule.
The invention also provides a host cell containing the expression vector.
The recombinant mannan-binding lectin ppg-rep-MBL of the present invention has low immunogenicity and can inhibit novel coronaviruses.
Research reports that (Pro-Hyp-Gly) 10 is a self-associated triple helix peptide with collagen molecular characteristics, many characteristics of assembly are similar to those of collagen fiber formation, .（Wuding Zhou, Self-association of Collagen Triple Helic Peptides into Higher Order Structures*[J]. Journal of Biological Chemistry, 2006, 281(44): 33-40）CPP(GPP)4GPR(GPP)4GC、CCPP(GPP)5GPR(GPP)5GCC、CCCPP(GPP)5GPR(GPP)5GCCC、CPP(GPP)4GPR(GPP)4GC、YCCPP(GPP)5GPR(GPP)5GCCY、CCPP(GPP)4GPR(GPP)4GCC、CCCPP(GPP)4GPR(GPP)4GCCC is a series of natural collagen-derived biological functional sequences, and the peptide consists of collagen-like triple helix peptide and has good gel forming property and rheological property. Wherein CPP (GPP) 4GPR (GPP) 4GC, CCPP (GPP) 4GPR (GPP) 4GCC, CCCPP (GPP) 4GPR (GPP) 4GCCC have low inflammatory properties, and with minimal inflammatory response elicited by CCCPP (GPP) 4GPR (GPP) 4GCCC, negligible sequence GEKGEP in .(Ichise, S. F. (2019). Development of a collagen-like peptide polymer via end-to-end disulfide cross-linking and its application as a biomaterial. Acta biomaterialia, 94, 361–371. ).MBL has been demonstrated to enhance phagocytosis of phagocytes in vivo by mediating binding to C1qR （Al-Refaei M A, Makki R M, Ali H M. Structure prediction of transferrin receptor protein 1 (TfR1) by homology modelling, docking, and molecular dynamics simulation studies[J]. Heliyon, 2020, 6(1): 523-528.）.
Therefore, the protein sequence which does not contain OGKXGP short motif and can normally form collagen-like structure is utilized for replacement, and on the premise that the collagen-like structure after replacement is similar to the original structure, the combination with MASP2 protein is ensured, MBL-MASP2 complex cannot be formed, so that downstream complement components are activated, and finally complement lectin pathway cannot be activated, so that the modified MBL can possibly not excessively activate complement system, cause inflammatory injury and even die when being used as a drug for treatment. The low immunogenicity MBL protein under the strategy transformation becomes a research and development break of the safety medicine, and the strategy is also a new strategy which is proposed by the inventor for the first time.
Compared with the recombinant mannan-binding lectin modified by CRD region disclosed in patent application No. CN201910560238.2, the recombinant protein ppg-rep-MBL of the mannan-binding lectin disclosed by the invention is selected to modify the collagen-like region of the recombinant protein ppg-rep-MBL so that the recombinant protein ppg-rep-MBL is not combined with MSAP2 protein, and thus the activation effect on complement lectin pathway is obviously weakened. Compared with the paper (influence of mannan-binding lectin on the function of human neutrophils, J South Med Univ, 2013, 33 (6): 842-846), the enhancement effect of ppg-rep-MBL of the modified protein of the invention on the stimulation of PMN to secrete TNF-alpha and IL-6 by candida albicans is obviously stronger than that of MBL, and can be inhibited by Mannan. In contrast to rhMBL, which was recombined in paper （Kaur S, Gupta V K, Thiel S, Sarma P U, Madan T. Protective role of mannan-binding lectin in a murine model of invasive pulmonary aspergillosis.[J]. Clinical and experimental immunology, 2007, 148 (2): 382-9）, ppg-rep-MBL activated complement to produce a significantly less deposition of C4b than rhMBL due to the inability to bind MASP 2. The invention tests the binding activity of MASP2 protein, S protein and mannans on ppg-rep-MBL, and finds that the ppg-rep-MBL does not bind with the MASP2 protein, has weakened activation effect on complement lectin pathway, simultaneously retains the binding capability with mannans and can specifically bind to S protein sugar chains. The experimental result of pseudovirus neutralization shows that the modified protein can effectively block infection of HEK293T-ACE2 cells by the novel coronavirus and can effectively neutralize the novel coronavirus.
The beneficial effects of the invention are as follows:
1. The modified protein disclosed by the invention is not combined with MASP2 protein, so that the activation effect on complement lectin pathway is obviously weakened, the combination property of the modified protein and mannose is maintained, and the modified protein can effectively combine and block the infection of novel coronavirus pseudovirus and Delta mutant strain thereof on HEK293T-ACE2 cells.
2. The novel MBL protein with greatly reduced activation and supplementation capacity and unchanged other biological activities has a great pushing effect on the aspects of resisting bacteria, resisting viruses, treating immune system diseases and the like, and further provides theoretical and technical support for creating a new generation of innovative drugs with antiviral property and low immunogenicity, and basic research support for reducing the direction of inflammatory response caused by new coronavirus infection through a complement system. Lays a foundation for the application research of the novel medicinal mannan-binding lectin with high efficiency and low toxic and side effects and the possible MBL defect diseases in the future, and simultaneously provides candidate possibility for the protein to be designed into the development of inhibitors or medicines with broad-spectrum anti-coronavirus infection.
Drawings
FIG. 1 is a model of MBL-CLR and ppg-rep peptide fragments respectively constructed by MODELLER4.0 software in example 1 (wherein, A. MBL-CLR region trimer homomodeling structure; B. Ppg-rep region trimer homomodeling structure);
FIG. 2 is a graph showing the analysis of hydrogen bonding at the interface between MBL-CLR and MASP2 in example 1;
FIG. 3 is a graph of hydrogen bond analysis of ppg-rep and MASP2 junctions in example 1;
FIG. 4 is a graph showing the concentration of ppg-rep-MBL protein measured by BCA in example 3;
FIG. 5 is a SDS-PAGE result of ppg-rep-MBL in example 3 (wherein A. Coomassie brilliant blue staining: M1: hi protein Maker; M2: pre-stained Maker; lane 1: non-reduced native MBL; lane 2: reduced native MBL protein; lane 3: non-reduced ppg-rep-MBL protein; lane 4: reduced ppg-rep-MBL protein; B. Western blot: maker: pre-stained Maker; lane 1: non-reduced ppg-rep-MBL protein; lane 2: non-reduced native MBL protein; lane 3: reduced ppg-rep-MBL protein; lane 4: reduced native MBL protein);
FIG. 6 is a graph showing the binding capacity of ELISA to detect ppg-rep-MBL protein to different pathogens in test example 2;
FIG. 7 is a graph of the glucose inhibition assay of ppg-rep-MBL protein in test example 3 (wherein A. Native MBL protein; B. Ppg-rep-MBL protein (< 0.001, p <0.01, p <0.05 VS TBS-Ca2+);
FIG. 8 is a graph showing the binding capacity of ELISA to detect ppg-rep-MBL and MASP2 in test example 4;
FIG. 9 is a graph showing the complement activation energy of the ppg-rep-MBL protein detected by ELISA in test example 5;
FIG. 10 is a graph showing the effect of ppg-rep-MBL protein on the regulation of PMN phagocytosis in test example 6;
FIG. 11 is a graph showing the effect of ELISA in test example 7 on the regulation of human peripheral blood neutrophil (PMN) secretion cytokines (wherein, the effect of A.ppg-rep-MBL protein on Candida albicans stimulated PMN secretion TNF-. Alpha.; the effect of B.ppg-rep-MBL protein on Candida albicans stimulated PMN secretion IL-6);
FIG. 12 is a graph showing the binding of ppg-rep-MBL to the trimeric S protein of SARS-CoV-2 (wherein, A. RMBL, analysis of binding of ppg-rep-MBL to the trimeric S protein (BLI); B. RMBL, binding of ppg-rep-MBL to the trimeric S protein (with/without PNGase F treatment)) in test example 8;
FIG. 13 is a graph of the binding of ppg-rep-MBL to the trimeric S protein of SARS-CoV-2Delta mutant (wherein, A. RMBL, analysis of binding of ppg-rep-MBL to the trimeric S protein of Delta mutant (BLI); B. RMBL, binding of ppg-rep-MBL to the trimeric S protein of Delta mutant (with/without PNGase F treatment)) measured by BLI in test example 8;
FIG. 14 is a graph of the evaluation of the anti-SARS-CoV-2 pseudovirus activity of rMBL ppg-rep-MBL in test example 9 (wherein A. Wild-type; B. Delta mutant).
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples were obtained commercially unless otherwise specified.
Example 1 determination of collagen substitution sequences and construction of a trimer Structure model and evaluation
(1) The MBL protein sequence was first downloaded from NCBI, and the MBL protein Model was constructed by replacing the MBL-CLR region sequence with the above sequence according to the series of artificial collagen sequences CPP(GPP)4GPR(GPP)4GC、CCPP(GPP)5GPR(GPP)5GCC、CCCPP(GPP)5GPR(GPP)5GCCC、CPP(GPP)4GPR(GPP)4GC、YCCPP(GPP)5GPR(GPP)5GCCY、CCPP(GPP)4GPR(GPP)4GCC、CCCPP(GPP)4GPR(GPP)4GCCC, reported in the literature using MODELLER4.0 and Swiss-Model, respectively, and the structural stability and reliability were evaluated. DOPE is a model evaluation program of atomic potential energy statistics, taking the credibility of different conformations between the same compound as a standard, and selecting the optimal conformation and score of each structure for comparison. The lower the number of MolPDFEnergy, DOPE score scores, the more reliable the result, the more stable the structure. The higher the GMQE, identity score, the higher the structure reliability and the closer the modeled structure is to the crystal resolution structure. The results are shown in Table 1, and the sequence CCCPP (GPP) 4GPR (GPP) 4GCCC replaced MBL-CLR, namely MolPDFEnergy, DOPE score and GMQE, identity of the MBL protein structure are all optimal, namely the protein structure after the sequence replacement is the most stable and the reliability is the highest. And the corresponding inflammatory response initiated by the sequence is proved to be the lowest sequence, so the sequence is selected for subsequent experiments.
TABLE 1 evaluation of protein Structure credibility following replacement of MBL-CLR by different collagen-like sequences
| Sequence name | Sequence (Sequence) | Probability Density function energy (Mol PDF ENERGY (kcal/Mol)) | Discrete optimized protein energy (DOPE Score) | Global Model Quality Evaluation (GMQE) | Consistency (Identity) |
| SEQ ID NO .5 | CPP(GPP)4GPR(GPP)4GC | 721.61 | -6263.27 | 0.55 | 52.15 |
| SEQ ID NO .6 | CCPP(GPP)5GPR(GPP)5GCC | 717.42 | -6109.53 | 0.56 | 54.32 |
| SEQ ID NO .7 | CCCPP(GPP)5GPR(GPP)5GCCC | 749.42 | -6461.18 | 0.57 | 56.18 |
| SEQ ID NO .8 | CPP(GPP)4GPR(GPP)4GC | 805.27 | -6719.85 | 0.53 | 47.61 |
| SEQ ID NO .9 | YCCPP(GPP)5GPR(GPP)5GCCY | 816.67 | -6864.20 | 0.51 | 46.15 |
| SEQ ID NO .10 | CCPP(GPP)4GPR(GPP)4GCC | 690.56 | -6018.10 | 0.63 | 60.25 |
| SEQ ID NO .11 | CCCPP(GPP)4GPR(GPP)4GCCC | 680.24 | -5969.61 | 0.66 | 72.73 |
(2) In order to make the length of the replaced MBL protein amino acid sequence more approximate to that of natural MBL protein amino acid sequence, when the above-mentioned selected collagen sequence is introduced, the PP 4GPR 4GPP 4G sequence is repeated, i.e. CCCPP GPP 4GPR 4G sequence is introduced, only N-terminal 3 cysteines are remained, and C-terminal 3 cysteines are removed, so that it can make N-terminal Cys make triple helix cross-linked by means of disulfide bond, at the same time does not change the bond-forming angle of C-terminal CRD region specific recognition sugar residue of MBL. And to ensure functional integrity of the engineered MBL protein, a motif sequence GEKGEP is maintained at the C-terminus of the sequence. The MBL-CLR area is replaced by CCCPP GPP 4GPR 4GGEKGEP, and the replaced MBL is named ppg-rep-MBL. The ppg-rep-MBL comprises three parts of an N end, a neck and a CRD of the MBL, and the amino acid sequence of the ppg-rep-MBL is shown as SEQ ID NO. 3.
(3) Further, modeling evaluation was performed on the protein structure before and after substitution. Models of MBL-CLR and ppg-rep peptide fragments were constructed by MODELLER4.0, respectively, and homology modeling was performed on the native MBL-CLR and ppg-rep peptide fragments using the BLAST program. And then respectively constructing the corresponding stable trimer structures of MBL-CLR and ppg-rep through MODELLER4.0, wherein the structural diagram is shown in figure 1. The ppg-rep protein body before and after replacement has a more regular trimeric collagen-like structure with most of the structure similar to that of the MBL-CLR region. The difference is that the N end of ppg-rep protein has a section of intertwining structure, some are embedded in the interior of the protein, and other are exposed on the surface of the external ppg-rep protein.
(4) Hydrogen bond analysis
In protein complexes, the presence of hydrogen bonds plays a key role in the stability of the complex, and the increase in hydrogen bonds enhances the affinity of active centers between proteins, the greater the number of hydrogen bonds, the more stable the complex. To assess the stability of the mock system, the number of hydrogen bonds at the binding interface between MASP2 and MBL-CLR, ppg-rep was analyzed, respectively.
At the junction of MASP2 protein with MBL-CLR, ppg-rep peptide fragments, the amino acid residues of the protein and bond lengths involved in hydrogen bond formation are listed in Table 1. It is generally believed that strong hydrogen bonds are present in the 2.4-2.7 a, medium strong hydrogen bonds are present in the 2.7-3.2 a, and weak hydrogen bonds are present in greater than 3.2 a. As can be seen from FIG. 2, there are 2 strong hydrogen bonds, 3 medium strong hydrogen bonds and 2 weak hydrogen bonds between the MASP2 protein and the amino acid residues at the binding interface of the MBL-CLR complex. Whereas in FIG. 3 there are a total of 3 weak hydrogen bonds greater than 3.2A at the MASP 2-ppg-rep binding interface, there are no strong hydrogen bonds and medium strong hydrogen bonds, and as a result it can be determined that the ppg-rep-MASP2 complex is less stable than the MBL-CLR-MASP2 protein complex.
EXAMPLE 2 construction and expression of purified ppg-rep-MBL
Step 1, a ppg-rep-MBL plasmid (synthesized by Beijing qing Ke biological Co., ltd.) constructed by a company is transformed into escherichia coli competent DH5 alpha, monoclonal is selected, the correctness of the recombinant plasmid is identified by a sequencing technology, and the sequencing work is completed by the Beijing qing biological Co., ltd. And selecting and verifying correct clone amplification culture extracted plasmids for standby.
Step 2. Secretory expression of ppg-rep-MBL protein using 293T cells. Insulin transferrin selenium (ITS, instrument-transferrin-Selenium) with concentration of 1% and ascorbic acid (vitamin C) with concentration of 60 μm/L are additionally added into DMEM culture medium, so that the expressed protein polymer structure is more stable. The ratio between cells, plasmid and transfection reagent was calculated according to the transfection reagent instructions, the plasmid in step 1 was transfected into 293T cells, after which the cells were placed in an incubator at 5% CO 2, 37℃for 72h, the cell supernatant containing ppg-rep-MBL protein was harvested and PMSF was added to one thousandth of the supernatant volume and placed at 4℃for temporary storage.
And 3, packaging the supernatant collected in the step 2 by using a dialysis bag (MD 44) with the aperture of 8-12 kDa, uniformly spreading PEG20000, concentrating until the volume is about 1/10 of the original volume, and taking out. The concentrated solution was separated and purified by using Mannan-Agarose protein purification column (10 ml), and the column was washed with 15-20 times of ultrapure water and 15 times of TBS-Ca 2+ buffer solution by column volume. The column was resuspended with 2 column volumes of TBS-Ca 2+ buffer and the ppg-rep-MBL protein sample Mannan-Agarose purification column was mixed in a ratio of 100. Mu.l column per 1ml sample. The protein and column mixture was placed on a vertical mixer and hung on the column overnight at 4 ℃. The next day, the mixture was removed and added to a 10ml column filter, the excess liquid was allowed to flow out of the filter column by gravity, the column was rinsed with TBS-Ca 2+ buffer, and the protein was washed out. The ppg-rep-MBL protein was eluted with 20mmol/LEDTA of TBS-Ca 2+ and collected in a centrifuge tube. The eluate was ultrafiltered with an ultrafiltration tube having a pore size of 30kDa, centrifuged at 4℃and 4000g for 10min, and the liquid inside the tube was retained. Dialyzing the ultrafiltered liquid at 20mmolTris-HCl and 150mmolNaCl (pH=7.4) dialysate overnight, and replacing the dialysate every 6-8h to obtain target protein.
Example 3 detection and concentration determination of ppg-rep-MBL after purification
Ppg-rep-MBL protein concentration was determined using the Biyundian BCA kit, while protein integrity was tested using SDS-PAGE and Western Blot.
As a result, the concentrations of ppg-rep-MBL and native MBL protein were 2.27mg/ml and 1.39mg/ml, respectively, as shown in FIG. 4, which was calculated according to the standard curve formula. The results of electrophoresis and WB are shown in FIG. 5, and the results of the coomassie blue staining show that in the non-reduced state, the relative molecular masses of the purified ppg-rep-MBL and natural MBL proteins (Mr is larger than 460kD, mainly in the form of pentamer and above), the rest of the proteins are more obvious in the vicinity of 32kD and 70kD, and exist in the form of ppg-rep-MBL, natural MBL single chain or double chains, in the reduced state, and exist in the vicinity of 32kD and 70kD in the form of single peptide chain or double peptide chain, and in the non-reduced state, by Western blot analysis, part of the proteins have molecular weights larger than 460kD and cannot be successfully transferred to PVDF membranes.
The beneficial effects of the invention are demonstrated by the following efficacy tests.
Test example 1 Yeast agglutination test to test the agglutination ability of ppg-rep-MBL protein
The obtained fresh saccharomycetes are centrifuged, washed 3 times with sterile TBS and resuspended, the density of the saccharomycetes is adjusted to be 1X 10 10 cfu/ml, the saccharomycetes are thermally inactivated for 1h at 65 ℃, and the saccharomycetes are cooled for standby. The native MBL protein, ppg-rep-MBL, was diluted to 20. Mu.g/ml with TBS-Ca 2+, diluted in a double ratio, added to the thrombin plate, and mixed with yeast. The reaction was carried out at room temperature for 1 hour using TBS-Ca 2+ as a negative control, and the results were observed and recorded under a microscope. The judgment criteria are as follows:
(1) Yeast was not coagulated, and was labeled (-) as the same as the negative control;
(2) 2 to 3 yeasts agglutinate, designated (+/-);
(3) 3 to 5 yeasts are aggregated and marked as weak positive (+);
(4) 5 to 10 yeasts are agglutinated and marked as positive (++);
(5) 10-20 intensive yeasts are aggregated and marked as (++);
(6) More than 20 dense yeasts are greatly agglutinated, is denoted as (+) ++).
TABLE 2 Yeast agglutination assay for the binding Activity of ppg-rep-MBL to native MBL protein
As a result of comparative analysis of the agglutination effect of purified ppg-rep-MBL with that of the native MBL protein, it was found from Table 2 that the agglutination titer of the purified ppg-rep-MBL protein was 1:512 and that of the native MBL protein was 1:512, at which time the concentrations of both proteins were 0.039. Mu.g/ml. The experimental results showed that the purified ppg-rep-MBL protein caused aggregation at a minimum concentration of 0.039. Mu.g/ml. That is, when the concentration of ppg-rep-MBL protein is less than 0.039. Mu.g/ml, the protein does not coagulate yeast any more, and compared with the natural MBL protein, the minimal coagulate concentration of the ppg-rep-MBL protein after modification is the same as that of the natural MBL protein, which roughly reflects that the activity of the ppg-rep-MBL protein is not affected before and after modification.
Test example 2 ELISA detection of the binding Capacity of ppg-rep-MBL protein to different pathogens
(1) Washing Candida albicans, saccharomycetes and Escherichia coli growing in logarithmic phase with PBS for 3 times, adjusting to 10 8 cfu/ml, diluting with a doubling ratio, adding an ELISA plate, 100 μl/hole, and coating at 4deg.C for 12h;
(2) The liquid in each well was aspirated and washed with TBS-T-Ca 2+, 300 μl/well, and the wash repeated 3 times;
(3) Spin-drying the washing liquid, and sucking residual liquid on clean water-absorbing paper;
(4) Naturally air-drying the ELISA plate at room temperature, adding 100 μl/hole of 4% paraformaldehyde, and fixing at room temperature for 30min;
(5) Repeating the steps 2-3, adding a sealing liquid, and sealing for 1h at room temperature, wherein the volume of the sealing liquid is 300 mu l/hole;
(6) Repeating the steps 2-3, diluting ppg-rep-MBL protein with TBS-Ca 2+, and incubating for 2 hours at room temperature at 20 mu g/ml per well;
(7) Repeating the steps 2-3, adding 100 μl/well of 1:2000 rabbit anti-human MBL monoclonal antibody (diluted with TBS buffer), taking the diluted solution as blank, covering with adhesive tape, and incubating at room temperature for 2 hr;
(8) Repeating the steps 2-3, adding 100 μl/well of HRP-goat anti-rabbit (diluted with TBS buffer) diluted 1:5000, incubating at room temperature for 20min, and avoiding placing the plate under direct light;
(9) Repeating the steps 2-3, adding the 1:1 mixed solution of A+B in the ELISA universal kit, and reacting for 20min at room temperature and in a dark place at 100 μl/hole;
(10) Add 50 μl of reaction stop solution to each well, tap the plate to ensure adequate mixing;
(11) The A450nm value was determined on a microplate reader within 15 min.
As a result, as shown in FIG. 6, ppg-rep-MBL protein was able to bind efficiently to pathogenic bacteria. But the binding capacity with 3 bacteria is different, wherein the binding capacity between ppg-rep-MBL protein and candida albicans and microzyme is stronger than that of the escherichia coli, and the escherichia coli only adsorbs a small amount of mannans in a culture medium on flagella because the candida albicans and microzyme cell walls contain a large amount of mannans, so that the binding capacity between the escherichia coli and ppg-rep-MBL protein is weaker than that between other two bacteria.
Experimental example 3 sugar inhibition experiment of ppg-rep-MBL protein
(1) Diluting Mannan to 20 mug/ml of coating liquid, adding the coating liquid to an ELISA plate, coating the ELISA plate for 12 hours at 4 ℃ with 100 mug/hole;
(2) The liquid in each well was aspirated and washed with TBS-T-Ca 2+, 300 μl/well, and the wash repeated 3 times;
(3) Spin-drying the washing liquid, and sucking residual liquid on clean water-absorbing paper;
(4) Adding a sealing liquid into the mixture, sealing for 1h at room temperature, 300 μl/well;
(5) Repeating the steps 2-3, adding 100 mu l of purified ppg-rep-MBL and natural MBL protein respectively, wherein the dilution of the experimental group only contains TBS-Ca 2+, and the dilution of the control group contains 20mmol/LEDTA, mannose, mannotriose and mannopyranose respectively, wherein the concentrations of the mannose, the mannotriose and the mannopyranose are 20 mu g/ml, and incubating for 1.5h at room temperature;
(6) Repeating the steps 2-3, adding 100 μl/well of 1:2000 rabbit anti-human MBL monoclonal antibody (diluted with TBS buffer), taking the diluted solution as blank, covering with adhesive tape, and incubating at room temperature for 2 hr;
(7) Repeating the steps 2-3, adding 100 μl/well of HRP-goat anti-rabbit (diluted with TBS buffer) diluted 1:5000, incubating at room temperature for 20min, and avoiding placing the plate under direct light;
(8) Repeating the steps 2-3, adding the 1:1 mixed solution of A+B in the ELISA universal kit, and reacting for 20min at room temperature and in a dark place at 100 μl/hole;
(9) Add 50 μl of reaction stop solution to each well, tap the plate to ensure adequate mixing;
(10) The A450nm value was determined on a microplate reader within 15 min.
As a result, as shown in FIG. 7, in FIG. 7A, the native MBL protein was able to bind efficiently to mannan (TBS-Ca 2+ group), but in other experimental groups, since saccharides such as mannose 3 and mannose 5 were present in the respective reaction systems, the binding of mannan to the native MBL protein was inhibited to various degrees by competing with mannan for binding to the sugar recognition domain (CRD) of the native MBL protein. Wherein, the EDTA group is provided because the natural MBL protein is Ca2+ -dependent protein, and EDTA is added in the dilution to chelate Ca 2+, so that the activity of the CRD site dependent on Ca 2+ in the natural MBL protein is lost, thereby inhibiting the combination of the natural MBL protein and mannan. Similarly, in FIG. 7B, the binding of the modified ppg-rep-MBL protein to mannan was also inhibited to a different extent, and the inhibition trend was similar to that of the native MBL protein, which fully demonstrates that the binding activity of the modified ppg-rep-MBL protein to different sugars was similar to that of the native MBL protein, and that the modification of the intact MBL gelatin region did not significantly affect the sugar binding activity.
Test example 4 ELISA detection of the binding Capacity of ppg-rep-MBL to MASP2
(1) The ppg-rep-MBL protein and the natural MBL protein obtained by purification are diluted from 20 mu g/ml to 0.625 mu g/ml by double ratio (TBS-Ca2+ dilution), an ELISA plate is added, 100 mu l/hole is coated for 12 hours at 4 ℃, and meanwhile, an uncoated hole is set as a blank control;
(2) The liquid in each well was aspirated and washed with TBS-T-Ca2+, 300 μl/well, and the wash repeated 3 times;
(3) Spin-drying the washing liquid, and sucking residual liquid on clean water-absorbing paper;
(4) Adding a sealing liquid into the mixture, sealing for 1h at room temperature, 300 μl/well;
(5) Repeating the steps 2-3, diluting MASP2 protein with TBS-Ca2+ to 20. Mu.g/ml, 100. Mu.l/well, and incubating at room temperature for 2h;
(6) Repeating the steps 2-3, adding 100 μl/well of 1:2000 rabbit anti-human MASP2 monoclonal antibody (diluted with TBS buffer), and incubating at room temperature for 2h;
(7) Repeating the steps 2-3, adding 100 μl/well of HRP-goat anti-rabbit (diluted with TBS buffer) diluted 1:5000, incubating at room temperature for 20min, and avoiding placing the plate under direct light;
(8) Repeating the steps 2-3, adding the 1:1 mixed solution of A+B in the ELISA universal kit, and reacting for 20min at room temperature and in a dark place at 100 μl/hole. To each well 50 μl of reaction stop solution was added and the plate was tapped to ensure adequate mixing and the A450nm value was measured on a microplate reader within 15 min.
Pictures were drawn using GRAPHPAD PRISM and as a result, as shown in figure 8, ppg-rep-MBL no longer has binding capacity for MASP2 compared to rMBL.
Test example 5 ELISA detection of complement activation Capacity of ppg-rep-MBL protein
(1) Adding Mannan with the concentration of 20 mug/ml and 100 mug/well into the ELISA plate, coating for 12 hours at 4 ℃, and setting the uncoated well as a blank control;
(2) The liquid in each well was aspirated and washed with TBS-T-Ca 2+, 300 μl/well, and the procedure was repeated 3 times;
(3) Spin-drying the washing liquid, and sucking residual liquid on clean water-absorbing paper;
(4) Adding a sealing liquid into the mixture, sealing for 1h at room temperature, 300 μl/well;
(5) Repeating the steps 2-3, diluting the purified ppg-rep-MBL and natural MBL protein with TBS-Ca 2+ from 20 mu g/ml to 0.625 mu g/ml, and incubating for 2 hours at room temperature;
(6) Repeating the steps 2-3, adding MBL defect serum diluted by 1:5, and incubating for 1h at 37 ℃;
(7) Repeating the steps 2-3, adding 100 μl/hole of 1:3000 rabbit anti-human C4b monoclonal antibody (diluted with TBS buffer), taking the diluted solution as blank, and incubating at room temperature for 2h;
(8) Repeating the steps 2-3, adding 100 μl/well of HRP-goat anti-rabbit (diluted with TBS buffer) diluted 1:5000, covering the plate and incubating at room temperature for 20min, and avoiding placing the plate under direct light;
(9) Repeating the steps 2-3, adding the 1:1 mixed solution of A+B, 100 μl/hole, and reacting at room temperature in dark place for 20min;
(10) Add 50 μl of stop solution to each well, gently tap the plate to ensure adequate mixing;
(11) The enzyme label instrument detects the A450nm value.
As a result, as shown in FIG. 9, mannans in the native MBL protein binding system can bind to MASP2 proteins in serum, allowing activation of MASP, resulting in cleavage of C4 to C4b, which activates complement. Whereas ppg-rep-MBL is unable to activate MASP because it is unable to bind to MASP2 protein, the ability of the final ppg-rep-MBL protein to activate complement is also significantly reduced (p < 0.001).
Test example 6 Regulation of PMN phagocytosis by ppg-rep-MBL protein
Step 1 preparation of fluorescein-labeled bacteria
(1) The logarithmic phase candida albicans is taken and adjusted to 1x10 9 cfu/ml.
(2) TBS was resuspended 3 times, resuspended in FITC solution dissolved in carbonate buffer, wrapped with tinfoil, and placed in 37 ℃ incubator for labeling 40min.
(3) TBS was washed three times and 4% paraformaldehyde was fixed for 30min.
(4) The TBS is washed twice, and the TBS is resuspended to a certain concentration and is reserved at 4 ℃.
Step 2 isolation of human peripheral blood neutrophils (PMNs)
The whole process is carried out at the temperature of 25+/-2 ℃ for samples, reagents and experimental environment.
(1) In a sterile siliconized centrifuge tube, 8ml of PMN separation solution was added.
(2) The sucked blood is gently dropped on the separated liquid surface, and centrifuged at 450g for 30min.
(3) Two layers of cyclic white cells appear in the tube, the upper layer being monocytes and the lower layer being PMN cells being aspirated.
(4) The aspirated PMNs were added to 8ml of wash solution and the cells resuspended.
(5) 300G centrifugation for 15min, the supernatant was discarded, the cells were resuspended in 6ml wash solution and centrifuged for 15min at 300 g.
(6) If there are residual erythrocytes, add erythrocyte lysate, wait for 3min.
(7) Centrifugation at 300g for 15min, discarding the supernatant, and repeating steps 7-8 until no red blood cells are visible to the naked eye.
(8) Washing 1-2 times, adding 2ml of 1 XPBS to resuspend cells, centrifuging for 6min at 350g, and discarding the supernatant.
(9) Neutrophil medium resuspended cells and the cells were counted.
(10) PMN counts were then placed in a 37 ℃ 5% co 2 cell incubator.
(11) Trypan blue dye staining verifies the activity of PMNs, white-Giemsa (Wright-Giemsa) dye staining observes cell morphology and flow analyses the purity of PMNs.
Step 3, mixing FITC marked candida albicans with human serogroup, natural MBL proteome and ppg-rep-MBL proteome, mixing with MBL defective serum according to a certain proportion, incubating for 30min at 37 ℃ in the presence of 5% CO 2 in the absence of light, adding PMNs (1X 10 6) and shaking for 30min at 37 ℃ in total 200 mu l/tube. TBS was washed 3 times, centrifuged at 600g for 10min, fixed with an equal amount of 4% paraformaldehyde, and the phagocytic effect was detected by flow cytometry, and the results were analyzed by FlowJo software. As shown in FIG. 10, in the group to which only the mixed human serum was added, the phagocytosis rate of neutrophils (PMN) against Candida albicans was only 12.2%, while the phagocytosis rate of the group to which the native MBL protein was added was 25.6%, and the phagocytosis rate of the group to which the ppg-rep-MBL protein was added was 23.5%, as compared with the mixed human serum group to which no protein was added, it was found that the ppg-rep-MBL protein was as much as the native MBL protein, and had an obvious promotion effect on Candida albicans phagocytosis by PMN, indicating that the modified ppg-rep-MBL protein still had a function of promoting the PMN to phagocytize pathogens.
Test example 7 ELISA the modulation of human peripheral blood neutrophil (PMN) secreting cytokines by ppg-rep-MBL was examined.
1X 10 5 cfu/ml Candida albicans and 4X 10 5 PMN cells were mixed and inoculated in 96-well plates, MBL-deficient serum was added at a final concentration of 5%, wherein one group was added with ppg-rep-MBL protein at a concentration of 0,2.5,5,10,20. Mu.g/ml, respectively, to determine the content of cytokine TNF-alpha, and the other two groups were added with ppg-rep-MBL protein at a concentration of 0,2.5,20. Mu.g/ml, respectively, and 100. Mu.g/ml Mannan was added to 20. Mu.g/ml ppg-rep-MBL protein for stimulation, and 3 replicates were set, 200. Mu.l/well, to determine the content of cytokines TNF-alpha and IL-6. Placing the cells in a cell culture box for continuous culture for 24 hours.
ELISA for detection of PMN secreted cytokines cell supernatant was collected, centrifuged at 2500g for 18min and the supernatant was collected with a fresh centrifuge tube. ELISA kit for human tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) is used for detecting the content of cytokines in PMN culture supernatant.
(1) Setting standard holes, and adding 50 μl of the diluted standard into each hole;
(2) Sample addition, namely adding 10 mu l of cell supernatant and 40 mu l of sample diluent into an ELISA plate for mixing;
(3) Adding enzyme-labeled reagent, namely adding 100 mu l of enzyme-labeled reagent into each hole, and adding no blank holes;
(4) Incubating, namely sealing the 96-well plate by using a sealing plate film at 37 ℃ and incubating for 60min;
(5) Washing liquid, diluting 20X washing liquid to 1X;
(6) Washing, namely pouring out liquid, spin-drying, adding 300 μl of washing liquid, pouring out after 30s, and repeating for 5 times;
(7) Color development, namely adding the mixed solution of A and B in a ratio of 1:1, and reacting for 20min at room temperature in a dark place with 100 mu l/hole;
(8) Stopping, namely adding 50 μl of stopping solution, and changing the blue color into yellow;
(9) Measuring, namely measuring the absorbance at 450nm within 10min after adding the stop solution;
(10) And establishing a standard curve, and calculating the content of cytokines in the sample.
The result of plotting GRAPHPAD PRISM on a graph shows that ppg-rep-MBL plays a dual role in Candida albicans infection, low concentration of ppg-rep-MBL has obvious promotion effect on secretion of TNF-alpha and IL-6 by PMN cells, and high concentration of ppg-rep-MBL inhibits cytokines secreted by the two PMNs, and the inhibition effect can be blocked by Mannan.
Test example 8 BLI detection of the binding Capacity of ppg-rep-MBL to SARS-CoV-2 Spike
Affinity detection of ppg-rep-MBL with S protein was performed using the biological membrane layer interference technique (Biolayer Interferometry, BLI). Affinity assays were performed using ForteBio Octet RED e and K2 systems (Pall ForteBio, USA). Ni-NTA biosensors (Pall forteBio, USA) were used for quantitative and kinetic characterization of his-tagged biomolecules. The sensor was pre-wetted in dialysis buffer for 15 minutes before use. The trimeric S proteins (S_Trimer and S_Trimer_Delta) and sugar-free chain trimeric S proteins (S_Trimer 'and S_Trimer_Delta') formed after co-incubation with PNGase F at 37℃were immobilized on the biosensor tip surface (20. Mu.g protein/1. Mu.L PNGase F, PBS 7.4), respectively, and each set of signal values was immobilized at 3.0 nm (balance: 100S; dissociation: 300S). ppg-rep-MBL at a concentration of 1. Mu.M was dissolved in PBS buffer (0.02% Tween 20). The trimeric S protein loaded biosensor was applied to the procedure of equilibration (PBS 7.4+0.02% Tween 20, 100S), binding (1. Mu.M ppg-rep-MBL, PBS 7.4+0.02% Tween 20, 180S), dissociation (PBS 7.4+0.02% Tween 20, 300S).
The binding or dissociation of the soluble ppg-rep-MBL-trimer S protein complex at the biosensor tip surface causes a mode shift of the reflected interference wave, which is expressed as a sensor map. The whole experimental procedure was carried out in a black 96-well microplate (Greiner Bio-One, austraia) at a temperature of 30℃and a rotational speed of 500 rpm, 200. Mu.L per well working volume. The protein S-loaded sensor was added to PBS buffer as a control to correct baseline drift. Normalization processing is performed on the response data by adopting Octet data analysis software (11.1.2.9 edition).
As a result, as shown in FIGS. 12 and 13, ppg-rep-MBL SARS-CoV-2 and its Delta mutant strain have slightly weaker binding ability to the trimeric S protein than rMBL, but are still able to bind efficiently to the S protein. And cannot be combined with the S protein with the sugar chains removed after PNGase treatment, so that ppg-rep-MBL plays a role in combination by combining the sugar chains on the surface of the S protein, namely, ppg-rep-MBL can be effectively combined with the sugar chains on the surface of the S protein.
Test example 9 neutralization Activity of ppg-rep-MBL on SARS-CoV-2 pseudovirus
(1) 293T cells (293T-ACE 2 cells) stably transformed with ACE2 plasmid were digested with pancreatin and then resuspended in complete medium [ DMEM Medium (Gibco) with 0.1. Mu.g/mL streptomycin (Thermo), 0.06. Mu.g/mL penicillin (Thermo), 10% w/v foetal calf serum (Excell Bio) ] to a concentration of 1X10 5/mL, plated in 96 well cell culture plates (Corning) at a density of 1X10 4 cells/well and incubated at 37 ℃.
(2) The protein samples to be tested (rMBL, ppg-rep-MBL) were diluted to a concentration gradient of 0.005-500. Mu.g/mL in double-free DMEM, ACE2 protein was diluted to a final protein concentration of 600ug/mL (about 4.6. Mu.M) in double-free DMEM and filtered with a 0.22 μm small filter for use. Equal titers of SARS-CoV-2 pseudovirus (including both the original strain and the Delta mutant, both available from Genomeditech) were diluted with double DMEM and a 0.08ul pseudovirus per well standard was added with 50ul pseudovirus-DMEM mixture per well.
(3) Mixing diluted pseudovirus with protein sample to be tested/ACE 2 protein at 1:1, mixing diluted pseudovirus with double DMEM at 1:1 (negative control group), incubating at 37deg.C for 1h, adding the protein sample to be tested/pseudovirus mixture into HEK293T-ACE2 cells, and replacing fresh complete DMEM medium after 24h of infection.
(4) After incubation at 37 ℃ for 48 h, luciferase activity in HEK293T-ACE2 cells was determined using One-LumiTM II firefly luciferase assay kit (Beyotime) and read by a fluorescent microplate reader. Finally, neutralization curves were plotted through GRAPHPAD PRISM 6 and median inhibitory concentration (IC 50) values were calculated. As a result, FIG. 14 shows that although the inhibitory effect of ppg-rep-MBL was reduced (IC 50 22.91.91. Mu.g/ml and 18.78. Mu.g/ml) as compared to rMBL on the novel coronavirus and Delta mutant pseudoviruses (IC 50 7.02.02. Mu.g/ml and 4.398. Mu.g/ml), the novel coronavirus and Delta mutant pseudoviruses were effectively neutralized.
Claims (3)
1. Use of the mannan-binding lectin-altering protein ppg-rep-MBL in the manufacture of a medicament for the treatment of coronavirus infection;
The amino acid sequence of the mannan-binding lectin-altering protein ppg-rep-MBL is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4;
the medicine is a medicine for blocking SRAS-CoV-2 infection.
2. The method of claim 1, wherein the SARS-CoV-2 comprises an original strain and a Delta mutant strain.
3. The method according to claim 1 or 2, wherein the method for preparing the mannan-binding lectin-altering protein ppg-rep-MBL comprises the steps of:
a. constructing recombinant manna binding lectin ppg-rep-MBL amino acid sequence by using wild manna binding lectin amino acid sequence SEQ ID NO. 1 as template and nucleotide sequence SEQ ID NO. 2, replacing the MBL-CLR region of the amino acid sequence of wild manna binding lectin with CCCPP GPP 4GPR 4GPP 4GGEKGEP, designing and synthesizing ppg-rep-MBL gene, the amino acid sequence SEQ ID NO. 3 and the nucleotide sequence SEQ ID NO. 4;
b. Constructing an engineered mannan-binding lectin recombinant plasmid by inserting ppg-rep-MBL gene between HindIII and XbaI restriction sites of vector pCMV3 to construct plasmid pCMV3-ppg;
c. the mannan-binding lectin recombinant protein ppg-rep-MBL was prepared.
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