CN102757503A - Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein - Google Patents

Abstract

The present invention provides human prostate apoptosis response protein 4 (Par4) or its selective apoptotic cancer cell area (SAC), and apoptin 2 ligand (Apo2L) fusion protein, namely Par4-Apo2L, SAC-Apo2L fusion Proteins, DNA sequences encoding these proteins, vectors containing these DNA sequences, host cells containing these vectors, methods for preparing these proteins by genetic engineering, and pharmaceutical compositions containing these proteins. The Par4, SAC and Apo2L of the present invention have the effect of selectively inducing tumor cell apoptosis, so the fusion protein can be used to prepare medicines for treating diseases such as tumors.

Description

Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein

technical field

The invention relates to the field of DNA recombination technology and biotechnology medicine. More specifically, the present invention relates to human prostate apoptosis response protein 4 (Par4) or its selective apoptotic cancer cell region (SAC), a protein fused with apoptin 2 ligand (Apo2L), a DNA encoding the fusion protein sequence, a vector containing the DNA sequence, a host cell containing the vector, a method for preparing the fusion protein by genetic engineering, and the application of the fusion protein in the treatment of diseases such as tumors.

Background technique

Par4 is an apoptosis-inducing gene first isolated from apoptotic prostate cancer cells by differential hybridization in 1994, so it is named: Prostate apoptosis response 4 protein (Par4)[ Cell Growth Differ, 1994;5(4):457-466.]. The Par4 gene is located on human chromosome 12q21.2 and consists of 7 exons and 6 introns. The coding sequence has 1023 nucleotides (SEQ ID NO.1), and the encoded Par4 protein consists of 340aa (SEQ ID NO.2). Par4 is now uniformly named internationally: PAWR (PRKC, apoptosis, WT1, regulator), and its IDs in major biological databases are: HGNC: 8614; Entrez Gene: 5074; Ensembl: ENSG00000177425; UniProtKB: Q96IZ0. In the protein deletion mutation analysis, it was found that the 59 amino acids consisting of 145-203aa of human Par4 are the core active region of apoptosis, which constitutes the minimum functional domain of human Par4 inducing apoptosis. This core region is the necessary and sufficient condition for Par4 to enter the nucleus, FasL/Fas localize to the cell membrane, activate the Fas-pro-apoptotic pathway, and inhibit NF-κB activity to induce apoptosis. This region can be located not only in the nucleus of tumor cells, but also in the nucleus of normal and immortal cells, but it only selectively induces the apoptosis of tumor cells. This shows that the induction of apoptosis by Par4 is realized by this core region. Since this core region does not induce apoptosis in normal cells, but can induce apoptosis in various tumor cells, it is called: selective for apoptosis induction cancer cells domain (SAC). Par4 and SAC can also selectively induce apoptosis of cancer cells outside the cell [Cell, 2009; 138(2): 377-388.]

In 1995, it was reported abroad that a gene encoding an anti-tumor protein was screened from a human expressed sequence tag (EST), and the protein encoded by the gene was called tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor). -related apoptosis-inducing ligand (TRAIL), also known as apoptotin-2 ligand (Apo2L), which can effectively induce apoptosis of tumor and transformed cells by initiating the intrinsic apoptosis program of cells, while for Normal cells are not affected. Apo2L is now uniformly named internationally: tumor necrosis factor (ligand) superfamily member 10 [tumor necrosis factor (ligand) superfamily, member 10], and its ID in major biological databases is: HGNC: 11925; Entrez Gene: 8743 ; Ensembl: ENSG00000121858; UniProtKB: P50591. The coding sequence of the human Apo2L gene has 846 nucleotides (SEQ ID NO.3), and the encoded Apo2L protein consists of 281 amino acids (SEQ ID NO.4). It has been proved that Apo2L is a typical type II transmembrane protein, and the extracellular region starting from the 95th or 114th position of its N-terminus can form free soluble apoptin 2 ligand protein, which also has the specific effect of initiating tumor apoptosis.

Since Par4 and Apo2L have different roles in inducing tumor apoptosis, the two are fused and expressed with a suitable amino acid linker [Protein Eng, 2003; 15(11): 871-879], and the resulting recombinant fusion protein It can not only inhibit tumors by selectively inducing tumor cell apoptosis through Par4, but also resist tumors by specifically initiating the intrinsic apoptosis program of tumor cells through Apo2L. Therefore, the fusion protein obtained by the present invention can enhance the anti-tumor effect under the synergy of the two, and can also reduce the trouble and pain caused by the separate administration of the two to patients, and provide new treatments for diseases such as anti-tumor. drug or preparation. Therefore, there is an urgent need in the art to develop new drugs that have both the biological activities of Par4 and Apo2L. In addition, there is an urgent need in this field to develop a production process with low cost and/or simple steps.

Contents of the invention

One object of the present invention is to provide a new drug with the biological activity of Par4 and Apo2L, which is the fusion protein of Par4 or its active fragment SAC and Apo2L.

Another object of the present invention is to provide DNA encoding the fusion protein, a vector containing the DNA sequence, and a host cell containing the vector.

Another object of the present invention is to provide a method for producing the fusion protein of Par4 or SAC and Apo2L with low cost and/or simple steps.

In a first aspect of the present invention, a fusion protein is provided, comprising:

(a) human prostate apoptosis response protein 4 (Par4) element, which has the amino acid sequence of human Par4 or its active fragment SAC;

(b) a human apoptin 2 ligand (Apo2L) element having the amino acid sequence of human Apo2L or an active fragment thereof; and

(c) Linker sequence of 0-20 amino acids located between the human Par4 element and the Apo2L element.

More preferably, the Par4 element has the amino acid sequence of 1-340 or 145-203 in SEQ ID NO.2;

The Apo2L element has the amino acid sequence of 95-281 or 114-281 in SEQ ID NO.4;

Moreover, the linker sequence is a linker peptide containing 4-14 amino acids;

More preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO.6 or 9.

In the second aspect of the present invention, there is provided an isolated DNA molecule encoding the above-mentioned fusion protein of the present invention. Preferably, the DNA molecule encodes a fusion protein comprising the amino acid sequence shown in SEQ ID NO.6 or 9. More preferably, said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO.5 or 7,8.

In the third aspect of the present invention, a vector containing the above-mentioned DNA molecule and a host cell containing the above-mentioned vector are provided.

In the fourth aspect of the present invention, a method for producing the fusion protein of the present invention is provided, which comprises the steps of:

Cultivating the above-mentioned host cells under conditions suitable for expressing the fusion protein, so as to express the fusion protein; and separating and purifying the fusion protein.

In the fifth aspect of the present invention, a pharmaceutical composition is provided, which includes a pharmaceutically acceptable carrier or excipient or diluent, and an effective amount of the fusion protein of the present invention.

In the sixth aspect of the present invention, the use of the fusion protein of the present invention is provided, which is used to prepare a drug for treating tumors.

Description of drawings

表示：Apo2L氨基酸序列；

表示：Par4氨基酸序列；......表示：氨基酸连接臂序列；表示：缺失部分氨基酸。Figure 1 shows the different splicing patterns of Par4, amino acid tether and Apo2L with different lengths. in:

Indicates: Apo2L amino acid sequence;

Indicates: Par4 amino acid sequence; ... Indicates: amino acid tether sequence; Indicates: missing some amino acids.

Figure 2 shows the electrophoresis analysis (SDS-PAGE) of a characteristic Par4-Apo2L fusion protein recombinant expression and purification process. Among them, A: the crushed supernatant of bacteria expressing Par4-Apo2L after 3 hours of induction; B: the crushed supernatant of bacteria not induced to express Par4Apo2L; C: the whole bacteria of Par4-Apo2L after induction of expression for 2 hours; D: after induction of expression for 1 hour Whole bacteria of Par4-Apo2L; E: metal-affinity-purified recombinant Par4-Apo2L fusion protein eluate; F: ammonium sulfate salting out active components of the expression supernatant; G: metal-affinity purified recombinant Par4-Apo2L fusion Protein activity peak; H: Par4-Apo2L further purified by S200; I: protein molecular weight marker (kD)

Figure 3 shows the electrophoresis analysis (SDS-PAGE) of a characteristic SAC-Apo2L fusion protein recombinant expression and purification process. Where A: protein molecular weight marker (kD); B: further purified SAC-Apo2L; C: activity peak of recombinant SAC-Apo2L fusion protein purified by metal affinity purification; D: eluate from metal affinity purification; E: heat-induced The broken supernatant of the expressed SAC-Apo2L engineering bacteria; F: the broken supernatant of the bacteria before the expression of SAC-Apo2L was induced

Detailed ways

After extensive and in-depth research, the inventors fused the Par4 coding sequence or its active fragment SAC coding sequence and the Apo2L coding sequence together to produce a fusion protein of Par4-Apo2L or SAC-Apo2L connected by a suitable amino acid linker. The fusion protein has the biological functions of both, and can selectively induce tumor cell apoptosis through Par4 or SAC, and can specifically initiate tumor cell apoptosis program to kill tumor through Apo2L. Therefore, the Par4-Apo2L or SAC-Apo2L fusion protein obtained in the present invention can enhance the anti-tumor effect under the synergy of the two, and can also reduce the trouble and pain caused by the separate administration of the two to the patient. Other treatments provide new compounds. The present invention has been accomplished on this basis.

definition

As used herein, the term "proapoptosis response protein 4 and apoptin 2 ligand fusion protein" and "Par4-Apo2L fusion protein" are used interchangeably, both referring to the amino acid sequence of the human prostate apoptosis response protein 4 element and A protein obtained by fusing amino acid sequences of human apoptin 2 ligands, wherein there may or may not be a connecting peptide sequence between the two. Furthermore, the fusion protein may or may not have an initial methionine or signal peptide.

As used herein, the term "prostate apoptosis response protein 4 element" and "Par4 element" in the fusion protein can be used interchangeably, referring to a part of the amino acid sequence in the fusion protein, which is similar to the natural or variant full-length human Prostate apoptosis response protein 4 or its active fragment SAC has substantially the same amino acid sequence, and has substantially the same biological activity as human natural prostate apoptosis response protein 4. The preferred Par4 element is human prostate apoptosis response protein 4, more preferably full-length human prostate apoptosis response protein 4 and its selective apoptotic cancer cell region, such as the 1-340th and The amino acid sequence of positions 145-203.

As used herein, the term "apoptin 2 ligand element" or "Apo2L element" in a fusion protein is used interchangeably to refer to a portion of the amino acid sequence in the fusion protein that is compatible with native or variant apoptin 2 ligand or a soluble active fragment thereof has substantially the same amino acid sequence and has substantially the same biological activity as native apoptin 2 ligand. A preferred Apo2L element is human apoptin 2 ligand, more preferably a soluble active fragment of human apoptin 2 ligand, such as the amino acid sequence at positions 95-281 or 114-281 in SEQ ID NO.4.

The sequences of prostate apoptosis response protein 4 and apoptin 2 ligands can be derived from humans or non-human animals, such as the amino acid sequences of human-derived SAC and mouse and rat-derived SAC are completely consistent Same. However, the human native sequence is preferred.

As used herein, the terms "linker peptide" or "amino acid linker" are used interchangeably and refer to the amino acid sequence located between the amino acid sequence of the prostate apoptosis response protein 4 element and the amino acid sequence of the apoptin 2 ligand element and the amino acid sequence of the SAC element. between short peptides or amino acids that act as linkers. The length of the connecting peptide is usually 0-20 amino acids, preferably 3-10 amino acids, most preferably 4-6 amino acids. The skilled person can follow conventional methods in this field [for example, see PNAS 1998; 95:5929-5934; Protein Eng, 2000; 13(5):309-312; Protein Eng, 2003; 15(11):871-879 and other documents] Design linker peptides. Usually, the connecting peptide does not affect or does not seriously affect the correct folding and spatial conformation of the amino acid sequence of the PAP4 element and the amino acid sequence of the apoptin 2 ligand element.

Preferred examples of connecting peptides include (but are not limited to): ① In order to facilitate protein folding into mutually independent structural domains, it is appropriate to use sequences such as GGGGSGGGGS as connecting arms, such as the 341-350th and 350th positions in SEQ ID NO.6 61-70 in SEQ ID NO.9; ② In order to facilitate the protease to cut Par4-Apo2L into two independent protein molecules, the enzymatic cleavage site IEGR of active factor X and the enzyme of metalloprotease with high tumor specific expression can be used The cleavage site PLGLWA is used as the linker. Similarly, the cleavage site of chymotrypsin 1, papain, plasmin, thrombin, trypsin and other enzymes can also be designed as the amino acid linker; ③ In order to facilitate purification, 6His can be used as a purification tag or as a Linker, to purify the fusion protein with metal affinity chromatography, similarly, maltose binding protein (MBP), glutathione-S transferase (GST), etc. can also be designed as a tag for purification or as a linker; ④ above The combination of the three schemes can also be designed into a new amino acid linker, such as the NVVVHQAHHHHHHEFTYK linker is a fusion of a protease cleavage site (NIa protease) and a metal affinity chromatography site 6His.

The DNA sequence encoding the fusion protein of the present invention can be completely artificially synthesized, can also be obtained by PCR amplification or synthesis, and can even be purchased from the market (such as the Par-4 coding sequence can come from Origene's Cat.No.SC110969; Apo2L coding sequence Cat. No. RC207596) from Origene), which are then spliced together to form a DNA sequence encoding the fusion protein of the present invention.

After obtaining the DNA sequence encoding the new fusion protein of the present invention, it is connected into a suitable expression vector and then transformed into a suitable host cell. Finally, the transformed host cells are cultivated, and the new fusion protein of the present invention is obtained by separation and purification.

As used herein, the term "vector" includes plasmids, cosmids, expression vectors, cloning vectors, viral vectors, and the like. Representative states include (but are not limited to): vectors that can be expressed in eukaryotic cells such as CHO and COS series, vectors that can be expressed in Saccharomyces cerevisiae or Pichia pastoris, and vectors that can be expressed in insect cells such as silkworm Vectors expressed in and prokaryotic expression vectors.

In the present invention, various vectors known in the art such as commercially available vectors can be used. For example, a commercially available vector is selected, and then the nucleotide sequence encoding the new fusion protein of the present invention is operably linked to the expression control sequence to form a protein expression vector.

As used herein, "operably linked" refers to the condition that some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, DNA encoding a signal peptide (secretion leader) is operably linked to the polypeptide DNA if the signal peptide is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, it is operably linked A ribosome binding site is operably linked to a coding sequence if it is placed in a position that enables its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells.

After the transformed host cell is obtained, the cell can be cultured under conditions suitable for expressing the fusion protein of the present invention, thereby expressing the fusion protein. Recombinant proteins can be isolated and purified by various separation methods taking advantage of their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include (but are not limited to): conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), Adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

In another aspect of the present invention, a pharmaceutical composition is also provided. The pharmaceutical composition of the present invention includes an effective amount of the novel Par-Apo2L or SAC-Apo2L protein of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient. In preparing these compositions, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or enclosed within a carrier in the form of a capsule or sachet. When the excipient acts as a diluent, it can be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Thus, the composition can be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions, and the like. Examples of suitable excipients include: lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl and propylparaben), sweeteners, and the like.

The compositions can be presented in unit or multiple dosage form. Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.

The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral or topical administration.

When using the pharmaceutical composition, a safe and effective amount of the fusion protein of the present invention or its antibody is administered to humans, wherein the safe and effective amount is usually at least about 1 microgram/kg body weight, and in most cases no more than about 10 mg/kg Body weight, preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

In addition, the fusion protein of the present invention can also be used in combination with other therapeutic drugs, including (but not limited to): various cytokines, such as IFN, TNF, IL-2, etc.; various tumor chemotherapy drugs, such as 5-Fu , methotrexate and other drugs that affect nucleic acid biosynthesis, nitrogen mustard, cyclophosphamide and other alkylating agents, doxorubicin, actinomycin D and other drugs that interfere with the transcription process and prevent RNA synthesis, vincristine, camptotheca Drugs that affect protein synthesis by bases, drugs that inhibit microtubules such as paclitaxel, and certain hormones and other drugs.

In summary, the main advantages of the present invention are as follows:

(1) Par4-Apo2L, SAC-Apo2L fusion protein not only has the function of Par4 or SAC to selectively induce tumor cell apoptosis, but also has the function of Apo2L to specifically initiate tumor cell apoptosis, and is a new drug for treating tumors .

(2) Convenient administration. Since the effective doses of the two are close in most cases, the simultaneous administration of the fusion protein facilitates the ratio of single drugs and reduces the pain of the subjects.

(3) Targeted. Par4 or SAC can transport Apo2L to the local tumor while selectively inducing tumor cell apoptosis; similarly, when Apo2L specifically acts on tumor cells, it can selectively induce tumor cell apoptosis by transporting Par4 or SAC to tumor tissue. effect of death.

(4) Enhance protein stability. After the two are fused and expressed, the half-life in vitro is significantly longer, and the stability in aqueous solution changes from about one week each to one to two months after fusion (both at 4°C); it is believed that the half-life in vivo will also be appropriate. extend.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention, not to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (NewYork: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1

Establish cDNA library and screen to obtain Par4 and Apo2L genes

1. Preparation of total RNA from human peripheral blood mononuclear cells

Separate human peripheral blood lymphocytes with lymphocyte separation medium according to routine, use RPMI-1640 medium (GIBCO company), add 10% newborn calf serum (Hangzhou Sijiqing biological company), penicillin and streptomycin and culture until adherent, The mononuclear cells were obtained, and then stimulated with 10 μg/L endotoxin of Escherichia coli R595 for 2 h to activate the mononuclear cells, collected ¹⁰ cells, and extracted total RNA with a total RNA extraction kit (Qiagen Company) to obtain human peripheral Total RNA of blood mononuclear cells.

2. Establish cDNA library and screen Apo2L gene

The total RNA obtained above was purified with an Oligo-dT column (Qiagen Company) to obtain total mRNA, and the cDNA synthesis kit (Clontch Company) was used to synthesize the first and second strands of cDNA according to the instructions, and then ligated after adding the EcoR I linker into a λgt10 vector, and packaged into a λ phage library using a λ phage packaging kit (Clontech Company), to construct a λgt10 cDNA library with a titer of 6×10 ⁶ .

The λgt10 cDNA library was plated on a 1×10 ⁵ colony/LB plate, and repeated impressions were made on a 20×20 cm nitrocellulose filter. Random primers were designed using the theoretical sequence of Apo2L, gene probes were prepared, and the library was hybridized and screened. Duplicate membranes containing colonies were plated in a plate screening buffer (50 mmol/L Tris-HCl pH ^7.5 , 1 mol/L NaCl, 0.1 % sodium pyrophosphate, 0.2% polyvinylpyrrolidone and 0.2% Ficoll) at 65°C for overnight hybridization. Plate screening at 65°C, washed twice with 2×SSC (0.3mol/L NaCl, 30mmol/L sodium citrate, pH7.0), and 0.1% SDS buffer. Then it was in close contact with the film at -40°C and screened for 40 hours.

Double positive samples were selected from the main plate to pick out the corresponding bacterium colony, and transferred to the LB plate of the buffer solution (100mmol/L NaCl, 10mmol/L MgSO ₄ , 50mmol/L Tris-HCl, pH 7.5) added with gelatin to obtain 12 positive plaques. 12 purified lambda DNAs were digested with Not I, electrophoresed on 1% agarose gel, and hybridized with the probe by Southern blotting. The clone with the highest radioactive intensity (about 1.7kp) was selected for DNA purification when hybridizing with the above probes. The inserts of these clones were subcloned into the Not I site of pSPORT1 (GIBCO). The DNA sequence of the insert in the plasmid is determined. From the results of sequence analysis, the insertion sequence in one of the plasmids is 1769bp, according to the translation initiation site requirements defined by Kozak, the 88th-90th nucleotide (ATG, encoding methionine) is the translation initiation site, Therefore, it contains a 5' untranslated region of 87bp, an open reading frame of 846bp and a 3' untranslated region (containing polyadenylation signal or polyA tail), and the coding sequence therein is shown as SEQ ID NO.3. Thus, the encoded 281 amino acid sequence is obtained as SEQ ID NO.4.

3. Screen Par4 gene

Using a method similar to steps 1 and 2, screen the human prostate cell cDNA library to obtain the coding sequence of Par4 such as SEQ ID NO.1, and the deduced encoded 340 amino acid sequence such as SEQ ID NO.2.

Example 2

The coding sequence of Par4 was obtained by reverse transcription-polymerase chain reaction (ie RT-PCR)

1. RT to obtain the first strand of cDNA of Par4

Using human prostate cell total RNA purchased from Clontech (USA) as a template and P1 as a primer, the first strand of Par4 cDNA was catalyzed by reverse transcriptase. Primers:

P1: 5'-CCA CAT TGA GTC TTG AAT CC-3' (SEQ ID NO.11)

2. Polymerase chain reaction (PCR) amplification to obtain the Par4 coding sequence

Using the above-mentioned first strand of cDNA as a template, P1 and P2 as primers, Tag DNA polymerase catalyzes the synthesis and amplification of the Par4 coding sequence. After sequence analysis, the obtained DNA sequence was consistent with the Par4 coding sequence displayed in various databases, that is, the Par4 coding nucleotide sequence was obtained. Primers:

P2: 5'-TAC AAG CTC CTC CAA GC-3' (SEQ ID NO.12)

Example 3

The coding sequence of Apo2L was obtained by RT-PCR

1. Preparation of total RNA from human peripheral blood mononuclear cells

(1) Separating lymphocytes from human peripheral blood according to conventional methods, and then obtaining mononuclear cells by the adherence method;

(2) The mononuclear cells were activated with bacterial endotoxin to stimulate the cells to express more genes and increase the abundance of RNA, and the total RNA was extracted with an RNA extraction kit (Qiagen Company).

2. Reverse transcription (RT) to obtain the first strand of cDNA of Apo2L

Using the above total RNA as a template and P3 as a primer, reverse transcriptase catalyzes the synthesis of the first strand of Apo2L cDNA. Primers:

P3: 5'-TCC AGG TCA GTT AGC CAA C-3' (SEQ ID NO.13)

3. Polymerase chain reaction (PCR) amplification to obtain the Apo2L coding sequence

Using the above-mentioned first strand of cDNA as a template, P3 and P4 as primers, Tag DNA polymerase catalyzes the synthesis and amplification of the Apo2L coding sequence. After sequence analysis, the obtained DNA sequence was consistent with the Apo2L coding sequence shown in various databases, and the Apo2L coding nucleotide sequence was obtained. Primers:

P4: 5'-CTG ACT TAC AGC AGT CAG-3' (SEQ ID NO.14)

Example 4

Combining artificial synthesis and PCR to obtain SAC coding sequence

1. Design and synthesis of SAC coding related sequences

According to the codon preference and codon degeneracy of Escherichia coli, each codon encoding SAC was designed and chemically synthesized in 4 stages by entrusting a biotechnology company (Shanghai Sangong). The characteristics of these four sequences are: P5 is a part of the SAC template strand, P6 is a part of the coding strand, and the 3' end sequences of P5 and P6 are complementary, that is, the underlined sequence in P5 and P6; The upper restriction endonuclease EcoR I enzyme cutting site, that is, the framed 6 nucleotide sequence in P7, optimizes the distance between the SD sequence and the foreign gene to 7 nucleotides (italics), and introduces the protein Synthetic start codon ATG, the 3' end sequence is the same as the 5' sequence of P5, that is, the sequence of the underlined wavy line in P7, and the SAC partial coding sequence in the middle; the 5' of P8 plus another restriction endonuclease BamH I restriction site, that is, the framed 6 nucleotide sequence in P8, the 3' end sequence is the same as the 5' sequence of P6, that is, the underlined sequence in P8, and the SAC partial coding sequence in the middle.

P5: 5'- GGC CAG ATT GAA AAA C GT AAA CTG CGT GAG AAA CGT CGCAGC ACC GGC GTG GTC AAC ATC CCG GCC GCA GAA TGC TTA GAT GAA -3' (SEQ ID NO. 15)

P6: 5'- AAT AGT GTT CTG TTG G GT AAT TGC ATC TTC ACG TTT ACGTTC TTT CTG GCC TGC TTC ATC ATC TTC GTA TTC ATC TAA GCA TTC -3' (SEQ ID NO. 16)

P7: 5'-AC ACA ATG CGT AAA GGC AAA GGC CAG ATT GAA AAA C -3' (SEQ ID NO. 17)

TTA AGC CTC ATT CTG AAT AGT GTT CTG TTG G-3’(SEQ ID NO.18)P8: 5'-AC

TTA AGC CTC ATT CTG AAT AGT GTT CTG TTG G -3' (SEQ ID NO. 18)

2. Polymerase chain reaction (PCR) amplification of SAC coding sequence

P5 and P6 serve as primers for each other to amplify part of the coding sequence of SAC by PCR. Then use the amplified product as a template, P7 and P8 as primers, PCR amplification again, the product is connected with T vector (TaKaRa company), and the DNA sequence analysis is consistent with the design, i.e. SEQ ID NO.19. Thus obtained the SAC coding sequence composed of E. coli preferred codons, and 5' and 3' with different restriction endonuclease sites, which can be used to construct pBV-SAC and construct SAC-Apo2L fusion in Example 5 PCR template for protein expression plasmids.

Example 5

Construction of expression vectors and engineering bacteria expressing fusion proteins

By rebuilding the vector pBV220, a recombinant plasmid for highly expressing SAC was constructed, and the constructed prokaryotic expression vector for expressing SAC was named pBV-SAC. The recombinant expression of the corresponding fusion protein of the present invention is carried out in the same way. The specific implementation steps are as follows:

1. Design and synthesis of oligonucleotide double strands

Synthetic oligonucleotide double strands, the sequence is as follows (the sequence of the complementary strand is not given):

P9: 5'-AA A GAT CT C TCA CCT ACC AAA CAA TGC CCC CCT GCA AAA AATAAA TTC ATA TAA AAA ACA TAC AGA TAA CCA TCT GCG GTG ATA AATTAT CTC TGG CGG TGT TGA CAT AAA TAC CAC TGG CGG TGA TAC TGA GCACAT CAG CAG GAC GCA CTG ACC ACC ATG AAG GTG ACG CTC TTA AAAATT AAG CCC TGA AGA AGG GCA GCA TTC AAA GCA GAA GGC TTT GGGGTG TGT GAT ACG AAA CGA AGC ATT GGT TAA AAA TTA AGG AG G AAT TC A CA-3'( SEQ ID NO.20)

Among them, the italic part is the _λPLPR tandem promoter sequence, the shaded part is the cI inhibitory protein binding site, the bold part is the optimized _SD sequence, the 5' end introduces the BglII restriction site AGATCT, and the 3' end introduces the EcoR I enzyme cutting site GAATTC;

P10: 5'-AA G GAT CC G TCG ACC TGC AGC CAA GCT TGG CTG TTT TGGCGG ATG AGA GAA GAT TTT C AG-3' (SEQ ID NO.21)

Among them, the BamH I restriction site GGATCC was introduced at the 5' end, followed by the Sal I, Pst I and HindIII restriction sites, and the Xmn I restriction site GAANNNNTTC was introduced at the 3' end.

2. Construction of expression vector pBV-SAC

The SAC coding sequence obtained by PCR amplification in Example 4 was digested with EcoR I and BamH I (the tool enzymes were all purchased from NEB, USA). Digest P9 with Bgl II and EcoR I, and digest P10 with BamH I and Xmn I. The pBV220 plasmid was digested with Bgl II and Xmn I.

Recover fragments of corresponding size from the agarose gel of the above enzyme-digested fragments, connect the fragments with T4 DNA ligase, and the resulting recombinant plasmid is the expression vector of the selective apoptosis cancer cell region, named pBV-SAC.

Compared with pBV220, pBV-SAC has the following characteristics: the SAC coding sequence is controlled by the upstream lambda phage _PLPR tandem promoter, _optimized ribosome binding sequence (SD sequence), inserts EcoR I restriction site and translation initiation codon At the same time, the distance between ATG and SD sequence was optimized to be 7 nucleotides, etc. This plasmid is the same as pBV220. It contains cIts857 binding sequence in the promoter sequence; it has ampicillin resistance gene; transcription termination sequence rrnB; the plasmid contains cIts857 gene encoding temperature-sensitive protein factor, and the encoded product is inactivated at 42°C. Thus, the repression of the promoter is lost, and the downstream foreign gene controlled by the promoter is expressed. The characteristic nucleotide sequences such as upstream regulatory sequence, optimized SD sequence, SAC coding sequence and translation termination sequence are shown in SEQ ID NO.10.

The SAC coding sequence can use the natural Par4 coding sequence obtained in Examples 1 and 2 as a template, and with the primers SEQ ID NO.23 and 25 listed in Table 1, PCR can obtain the natural SAC coding sequence, and then use the above-mentioned same method, pBV-SAC can also be constructed. Although the resulting recombinant plasmids encode different SAC sequences, the encoded product sequences are identical, i.e. positions 145-203 in SEQ ID NO.2.

3. Each fragment is introduced into a restriction site to construct a fusion protein expression vector

Using the same method as above, use the sequences listed in Table 1 as primers (the listed primers only partially enumerate the amplification of the 8 fusion proteins listed in Figure 1), use the above-mentioned examples or commercially available (such as the Par-4 coding sequence can be From Origene's Cat.No.SC110969; Apo2L coding sequence can come from Origene's Cat.No.RC207596) The corresponding coding sequence obtained as a template, PCR amplification of the corresponding coding DNA fragments, the corresponding restriction endonuclease digestion The PCR product and the tether coding sequence (flexible amino acid tether) formed by SEQ ID NO.38, 39, T4 DNA ligase is connected into the pBV220 vector of the above-mentioned transformation that has been digested with restriction endonuclease EcoR I and Pst I, The competent cells are transformed, the clones containing the recombinant plasmids are screened, and the DNA sequence determination confirms that the expression engineering bacteria expressing each fusion protein can be obtained.

Similarly, it is also possible to use SEQ ID NO.40, 41 to encode the highly expressed metalloprotease cleavage site coding sequence as the amino acid linker to construct the expression engineering bacteria of each fusion protein.

4. Overlap PCR to construct fusion protein expression vector

Similarly, SEQ ID NO.42, 43 and 44, 45 can be used as overlapping PCR primers, and the SAC obtained in Example 4 and the Apo2L coding sequence obtained in Example 1, 2 or the commercially available Apo2L coding sequence can be used as templates, and other corresponding Primers, PCR amplification once, can get the coding sequence of SAC-Apo2L fusion protein with flexible amino acid or metalloprotease cleavage site highly expressed in tumor as amino acid linker, and then digest with EcoR I and Pst I, T4 The DNA ligase is ligated into the same enzyme-cut vector, the competent cells are transformed, the clone containing the recombinant plasmid is screened, and the DNA sequence determination confirms that the expression engineered bacteria expressing the fusion protein can be obtained.

Similarly, other fusion protein expression engineering bacteria can also be constructed in the same way. Similarly, fusion proteins connected by other amino acid linking arms are also constructed in the same way.

Obviously, compared with the restriction endonuclease cutting sites introduced above, the fusion protein constructed by overlapping PCR lacks 4 amino acids encoded by 2 restriction endonuclease cutting sites. But the amino acid tether sequence is the same.

Table 1 Primer Nucleotide Sequence List

The first group: Par4 in the amplified fusion protein N-terminus of the fusion protein, as shown in Figure 1 A-D (p represents primer, P represents using embodiment 1, 2 or commercially available Par4 as a template, A represents using Example 1, 2 or commercially available Apo2L as a template, S indicates the SAC synthesized in Example 4 as a template, the middle number indicates the start or end position of the encoded amino acid, f indicates the forward primer, r indicates the reverse primer )

name sequence serial number Restriction sites P1f 5'-at gaa ttc aca atg gcg acc ggt ggc tac c-3' SEQ ID NO.22 EcoR I pP145f 5'-gc gaa ttc aca atg agg aaa ggc aag ggg c-3' SEQ ID NO.23 EcoR I pS 5'-gc gaa ttc aca atg cgt aaa ggc aag ggc c-3' SEQ ID NO.24 EcoR I Common to pP203r and pS59r 5’-gc gga tcc agc ttc att ctg aat ag-3’ SEQ ID NO.25 BamH I pP340r 5’-aa gga tcc cct ggt cag ctg acc c-3’ SEQ ID NO.26 BamH I pA95f 5’-tc gtc gac acc tct gag gaa acc att tc-3’ SEQ ID NO.27 Sal I pA114f 5’-aa gtc gac gtg aga gaa aga ggt cct c-3’ SEQ ID NO.28 Sal I pA281r 5’-ccg ctg cag tta gcc aac taa aaa ggc-3’ SEQ ID NO.29 Pst I

The second group: amplify Apo2L in the encoded fusion protein at the N-terminus of the fusion protein, as shown in Figure 1 a-d (in order to distinguish the first group of primers, add 1 after the primer name shown)

The third group: the introduction of amino acid linker

^* Corresponding f and r can form oligonucleotide double strands with sticky ends with restriction sites through simple denaturation and renaturation treatment, which are used to introduce restriction sites for each fragment to construct fusion protein expression vectors

A series of recombinant plasmids expressing Par4-Apo2L fusion proteins with different lengths and connection modes were thus obtained. In particular, the eight Par4-Apo2L recombinant fusion proteins prepared with the listed primers as shown in Figure 1 can directly exhibit the biological effects of killing tumor cells in vitro and inhibiting the growth of transplanted tumors in vivo.

A preferred nucleotide sequence for expressing the Par4-Apo2L fusion protein is shown in SEQ ID NO.5, and the deduced amino acid sequence is shown in SEQ ID NO.6.

Another preferred nucleotide sequence for expressing the SAC-Apo2L fusion protein can be as shown in SEQ ID NO.7, or as shown in SEQ ID NO.8 of SAC codon optimization, and their deduced amino acid sequences are consistent , as shown in SEQ ID NO.9.

So far, all nucleotide or amino acid sequences involved in the present invention have been mentioned, see the list of nucleotide or amino acid sequences. The sequence listing numbers are described as follows:

SEQ ID NO.1: Par4 encoding nucleotide sequence

SEQ ID NO.2: Par4 amino acid sequence

SEQ ID NO.3: Apo2L coding nucleotide sequence

SEQ ID NO.4: Apo2L amino acid sequence

SEQ ID NO.5: Par4-Apo2L coding nucleotide sequence

SEQ ID NO.6: Par4-Apo2L amino acid sequence

SEQ ID NO.7: Native SAC-Apo2L coding nucleotide sequence

SEQ ID NO.8: Synthetic SAC-Apo2L coding nucleotide sequence

SEQ ID NO.9: SAC-Apo2L amino acid sequence

SEQ ID NO.10: pBV-SAC characteristic nucleotide sequence

SEQ ID NO.11-45: Nucleotide sequences of primers and adapters

Example 5

Transform Escherichia coli to establish engineering bacteria

A series of recombinant expression plasmids such as pBV-SAC obtained in Example 4 were transformed into Escherichia coli BL21 [genotype: hsdS gal (λcIts857ind1 Sam7 nin5 lacUV5-T7)] (available from Shanghai Sangong Bioengineering Co., Ltd.) as usual, from Plasmid DNA was isolated from ampicillin-resistant colonies, identified by enzyme digestion, and confirmed by sequencing. The positive clones obtained were engineering bacteria expressing corresponding proteins.

Example 6

Preparation of Par4-Apo2L fusion protein

Various media are as follows: LB medium is used for test tube seed culture, 2×YT medium is used for secondary seed culture, semi-synthetic medium is used for fermentation, and fed batches are added to culture.

LB medium formula (g/L): peptone: yeast powder: NaCl=10:5:5;

2×YT medium formula (g/L): peptone: yeast powder: NaCl=16:10:5;

Formula in semi-synthetic fermentation medium (g/L): peptone: yeast powder: KH ₂ PO ₄ : K ₂ HPO ₄ : Na ₂ HPO ₄ .12H ₂ O: (NH ₄ ) ₂ SO ₄ : NH ₄ Cl=5 :5:2:4:7:1.2:0.2;

Various trace element solutions, concentration (g/L): MnSO ₄ .5H _{2 O:} CaCl ₂ .6H ₂ O: _{Na 2} MoO ₄ .2H 2 O _: ZnCl ₂ : CuSO ₄ .5H ₂ O: H ₃ BO ₄ :FeSO ₄ .7H ₂ O:CaCl ₂ .2H ₂ O:MgSO ₄ .7H ₂ O=0.001:0.004:0.002:0.002:0.001:0.0005:0.02:0.02:0.3;

Fermentation feed formula (g/L): glucose:yeast powder:peptone:MgSO ₄ .7H ₂ O=200:70:70:5.7.

The pH of the medium was adjusted to 7.2 throughout. After high-temperature sterilization of various media, ampicillin was added to a final concentration of 100 μg/ml.

After the primary seeds are cultivated overnight, they are transferred to the secondary seeds at a ratio of 1:20-100, and the fermenter is inoculated into the secondary seeds cultivated overnight according to 1-5% of the working volume. The cultivation is carried out in two stages, 5-7 hours at 32°C; 4-5 hours at 42°C. The constant flow pump adds feed, and the dissolved oxygen is controlled between 30-50%. Wet cells of about 30-100 g/L fermentation broth can be obtained.

Sonicate the fermented cells, centrifuge to take the supernatant, pass through a 0.22 μm filter membrane; perform affinity chromatography with metal affinity chromatography columns such as NTA Supperflow (Qiagen Company) or TALON Metal Affinity Rsins (Clontech Company), 5-10 mmol /L imidazole, pH 7.0 to elute the impurity protein, and then 80-100mmol/L imidazole, pH 7.0, to elute the recombinant protein; the eluate was passed through CM cellulose to collect the active components; and further purified by Sephacryl S-200 , and high-purity recombinant fusion protein can be obtained.

Results: Taking the prokaryotic recombinant expression of the two fusion proteins (Par4-Apo2L and SAC-Apo2L) shown in Figure 1-B and D as an example, the engineered bacteria were induced to express by heat, and separated by centrifugation after sonication. SDS-PAGE was performed on the supernatant of the supernatant, and it can be seen that the expressed fusion protein accounted for more than 10% of the total protein of the supernatant, and it was electrophoresis pure after further purification. The results are shown in Figures 2 and 3. Similar results can be obtained after induction and expression of the remaining individual Par4, SAC, Apo2L or fusion of the two, and recombinant proteins with corresponding molecular weights can be obtained.

Example 7

Determination of the biological activity of the fusion protein

In this example, the Apo2L activity and Par4 activity of the fusion protein were measured.

Apo2L activity: In vitro, human pancreatic ductal carcinoma 1990 cells, human large cell lung cancer NCI-H460, mouse melanoma B16, etc. can be used as target cells to determine the biological activity of Apo2L, and in vivo to inhibit human colon cancer in nude mice HCT-8 xenografts and other mouse xenografts that are sensitive to Apo2L in vitro and can form tumors in vivo were used as models to verify the biological function of Apo2L.

Par4 activity: In vitro observation of Par4 can target human large cell lung cancer NCI-H460, human prostate cancer PC-3, mouse melanoma B16, etc., to determine the biological activity of Par4, and in vivo to inhibit human colon cancer HCT in nude mice -8 xenograft tumors and other mouse xenograft tumors that are sensitive to Par4 in vitro and can form tumors in vivo were used as models to verify the biological function of Par4.

The specific test operation is as follows:

1. Analysis of Par4 and Apo2L activity: killing various tumor cells

Various target cells were obtained from the Institute of Cells, Chinese Academy of Sciences or Shanghai Changhai Hospital. Mainly human pancreatic ductal carcinoma 1990, 8898 strains, human large cell lung cancer NCI-460, human colon cancer HCT-8, human gastric cancer M85 strain, human neuroblastoma SK-N-SH strain, human laryngeal carcinoma Hep-2 strain , human nasopharyngeal carcinoma CNE-2 strain, human endothelial cell CEV304 strain, human fibroblast cell line, human colon cancer AT-29, human ovarian cancer 3AO, mouse fibroblast L929 strain, human glioma U251, human breast cancer, Human liver cancer HepG-2, SMMU7721, human hematological tumors (U937, Jukart, HL60, etc.), melanoma B16-MB, Ehrlich ascites tumor, Lewis sarcoma, etc.

Taking H460 cells as an example, seed cultured cells into 96-well cell culture plates at 2× ¹⁰⁸ cells/L, incubate in a 5% CO ₂ incubator at 37°C for 4-6 hours, discard the supernatant; Add to the cell plate after serial dilution; observe the effect of adding 1.5 μg/ml actinomycin D on the killing cells of various factors at the same time; the definition of activity unit: make 50% of the cells in the well of the culture plate die as a unit, and the titer That is, the reciprocal of the dilution of the sample at which 50% killing is achieved. Quantitative analysis by crystal violet method: discard the supernatant of adherent cells, stain with crystal violet fixative (5g/L crystal violet, 80mL/L formaldehyde, 1g/L NaCl, 200mL/L ethanol) for 15 minutes, wash away crystal violet with distilled water , dried, and the stained cells are living cells. Add 200 μL of 330 mL/L acetic acid to each well, rotate the shaker to dissolve the crystal violet, set the enzyme-linked analyzer to read the A value at a wavelength of 595 nm, and use the blank well without cells as the A background. Suspension cells were determined by the MTT method to determine the number of viable cells. The activity titer is linearly regressed with the logarithm of sample dilution and the mean of A595nm to obtain constants A and B, and (A control-A background)/2 is the 50% killing end point (Y). Calculate the activity unit of the sample according to Y=A+BlgX, that is, the X value. At the same time, the cells showed a typical apoptotic morphology under the action of Par4, or Apo2L, or the fusion protein. For example, under the action of 10 μg/L apoptin 2 ligand, typical changes in the cells were observed within 2 hours.

2. Fusion protein inhibits transplanted tumors in mice

Expansion of various tumor cells in vivo and in vitro: The tumor cells cryopreserved in liquid nitrogen were revived and cultured or inoculated into the peritoneal cavity of Kunming mice (10 ⁷ cells/mouse). After 10 days, the mice were sacrificed, and the ascites fluid of the mice was aspirated under aseptic conditions, and the cell concentration was adjusted to 10 ⁸ /ml with PBS. 0.2 ml of the cell suspension was inoculated subcutaneously in experimental mice. Randomly grouped, 6 mice in each group, subcutaneously inoculated mice were given 0 (PBS, control group), 10, 100, 1000 μg (sample)/kg (mouse body weight) respectively, administered every other day for 2 weeks. One week after drug withdrawal, the mice were sacrificed, the tumors were peeled off and weighed, the average tumor weight and tumor inhibition rate of each group were calculated, and the results were analyzed. The tumor inhibition rate was calculated by the following formula:

Results: Table 2 shows the results of transplanted tumors of human colon cancer cell HCT-8.

Table 2 HCT-8 test result table

It can be seen from Table 2 that the recombinantly expressed fusion protein has a better inhibitory effect on colon cancer xenografts.

Similarly, for other transplanted tumors, such as human large cell lung cancer NCI-H460, human pancreatic cancer SW1990, 8898, human gastric cancer M85, human laryngeal cancer Hep-2, human nasopharyngeal cancer CNE-2, human colon cancer AT-29, human Similar results can be obtained in glioma U251, human liver cancer HepG-2, melanoma B16-MB, Ehrlich ascites tumor, Lewis sarcoma and other models.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A fusion protein, characterized in that it consists of the following elements: (a) human prostate apoptosis response protein 4 (Par4) element, which has the amino acid sequence of human Par4 or its active fragment-selective apoptotic cancer cell region (SAC), that is, the first one in SEQ ID NO.2 - the amino acid sequence at position 340, or at position 145-203; (b) human apoptin 2 ligand (Apo2L) element, which has the amino acid sequence of human apoptin 2 ligand or an active fragment thereof, that is, 95-281 or 114-281 in SEQ ID NO.4 The amino acid sequence of the position; and (c) Linker sequence of 0-20 amino acids located between the human Par4 element and the Apo2L element. 2. The fusion protein according to claim 1, wherein the linking sequence contains 4-14 amino acids. 3. fusion protein as claimed in claim 1, is characterized in that, described fusion protein has the amino acid sequence shown in SEQ ID NO.6,9. 4. An artificially synthesized DNA molecule, characterized in that it encodes the fusion protein according to claim 1. 5. DNA molecule as claimed in claim 4, is characterized in that, it has the nucleotide sequence shown in SEQ ID NO.5,7,8,10. 6. A vector comprising the DNA molecule of claim 4. 7. A host cell, characterized in that it contains the vector according to claim 6. 8. A method for producing the fusion protein according to claim 1, characterized in that the steps are as follows: (1) set up a cDNA library, and screen to obtain Par4 and Apo2L genes; (2) RT-PCR obtains the coding sequence of Par4 and the expression of Apo2L Coding sequence, or chemically synthesize the coding sequence of SAC with Escherichia coli codon preference; (3) construct fusion protein expression vector; (4) transform Escherichia coli, establish engineering bacteria; (5) prepare fusion protein; (6) fusion The biological activity of the protein is determined. 9. A pharmaceutical composition, characterized in that it consists of a pharmaceutically acceptable carrier or excipient or diluent, and an effective amount of the fusion protein according to claim 1. 10. The use of the fusion protein according to claim 1, characterized in that it is used for the preparation of medicaments for treating tumors.

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