CN1696155A - The gene and its coded polypeptide inhibit the activation of NF-kB and NFAT - Google Patents

Abstract

A human gene NFIF1 for suppressing the activation of NF-kB and NFAT, its cDNA sequence, the polypeptide coded by it, the process for testing its functions by dual-leuciferinase report gene method, and its application in preparing the medicines for preventing and treating inflammation, anaphylactia, autoimmunopathy, tumor etc are disclosed.

Description

Genes and their encoded polypeptides that inhibit the activation of NF-κB and NFAT

technical field

The invention relates to the field of gene expression regulation, in particular to a gene that inhibits the activation of human nuclear factor NF-κB and NFAT, a preparation method of the gene, an expression vector containing the gene, a polypeptide encoded by it, an antibody to the polypeptide, and the polypeptide , antibodies in the prevention and treatment of diseases related to human nuclear factor NF-κB and NFAT, especially drugs for inflammation, allergic diseases, autoimmune diseases and tumors, and in the development of drugs that regulate lymphocyte activation, proliferation and apoptosis use.

Background technique

NFAT and NF-κB proteins are members of the transcription factor superfamily, and although they are expressed in many types of cells, they play key roles in regulating the activation, proliferation and activation-induced cell death of lymphoid cell lineages. NFAT and NF-κB have many commonalities, including similar DNA binding regions, rapid nuclear translocation after antigen stimulation, and so on. They overlap and antagonize each other at the same time, and they constitute a fine gene expression regulation network system in lymphocytes. NF-κB regulates innate and adaptive immune responses, while NFAT regulates adaptive immune responses. There are obvious differences in the functions of NFAT and NF-κB. NFAT regulates cell death induced by T/B cell activation, while NF-κB protein often plays a strong anti-apoptotic effect in lymphocytes and other cells. The anti-apoptotic activity of NF-κB contributes to their oncogenic potential, while the pro-apoptotic activity of NFAT makes it a tumor suppressor in lymphocytes.

NFAT and NF-κB proteins have similar DNA binding domains (about 300 Aa). NF-κB is the Rel functional domain, and NFAT is the RSD functional domain. Although the RSD and Rel functional domains have only 20% sequence homology, they both have 10 B sheets and 2 loop structures at the same position, which are used to bind similar DNA sequences and interact with other transcription molecules such as AP-1 role (Chen, Glover, Hogan, Rao, & Harrison, 1998).

Nuclear factor NF-κB is one of the most important transcription factors in the human body. It is involved in inducing the expression of various cells and viral genes, and plays a pleiotropic role in the regulation of gene expression in various types of cells. These genes are activated in response to inflammatory, immune and apoptotic signals and include many cytokines, chemokines, receptors, adhesion molecules, etc. NF-κB is also involved in the initiation of transcription from viral promoters, such as human immunodeficiency virus type I (HIV-1).

Mammalian NF-κB contains 5 proteins: p65 (RelA), p50 (NF-κB-1), RelB, c-Rel, p52 (NF-κB-2, p50B), which have 300 amino-terminal regions Amino acid similarity is called the Rel homology region. The five NF-κB protein molecules can form homologous or heterologous dimers, bind to specific DNA recognition sites with different binding specificities, and have different transactivation activities.

Since the activation of NF-κB can promote the transcription and expression of many pro-inflammatory factors, the excessive activation of NF-κB can cause inflammation, allergic diseases and autoimmune diseases; because the activation of NF-κB can promote cell growth and inhibit apoptosis Therefore, it plays an important role in the occurrence and development of tumors; in addition, the activation of NF-κB can block the apoptosis of some cells, so it also has an important function in regulating the growth and differentiation of cells.

TNFα (Tumor Necrosis Factor α), PMA (Myristate), IL-1 (Interleukin-1), LPS (Lipopolysaccharide) can all promote NF-κB activation; MEKK (mitogen-activated protein kinase kinase), TRAF (tumor necrosis factor receptor-associated factor) and other NF-κB signaling pathway positive regulatory genes can also promote NF-κB activation; while IL-10 (interleukin-10), FK506 (Fujita Sodium), glucocorticoids, aspirin, etc. can significantly inhibit the activation of NF-κB.

In view of the important role played by NF-κB in regulating human physiological functions and in the occurrence and development of various diseases (Burke J.R.Curr Opin Drug Discov Devel.20036(5), 720-8), inhibitors of NF-κB and suppressor genes will have important applications in the treatment of inflammatory diseases and tumor diseases. At present, the field of NF-κB activation regulation has become a focus of basic medicine, clinical medical research and drug development, such as I-KappaB-α, I-KappaB-β and other regulatory proteins related to NF-κB activation (US Patent No. 5,597,898). In addition, many negative regulators of NF-κB such as anti-interleukin-1 therapy, anti-tumor necrosis factor therapy, hormone therapy, aspirin, non-hormonal anti-inflammatory drugs, etc. (refer to the review article Nature Review Immunology, 2002, 2: 725; J Clin Invest, January 2001, Volume 107, Number 2, 135-142) have been widely used clinically, but these drugs have the disadvantages of weak specificity, insufficient inhibition and insufficient stability, and There are often side effects. Therefore, it is necessary to further explore new regulatory genes that inhibit NF-κB activation, and to study the regulatory function of its expression in cells on NF-κB activation, so as to develop therapeutic drugs for inflammation, allergic diseases, autoimmune diseases and tumors. Drugs provide the necessary basis.

NFAT is a multifunctional transcriptional molecule that is expressed in most immune cells and plays a central role in regulating the expression of a range of cytokines and cell surface ligands that play a key role in regulating immune Response plays a key role. Stimulate the antigen receptors of immune cells, trigger the influx of calcium ions, and generate activated calcineurin (CaN), which catalyzes the dephosphorylation of NFAT proteins, thereby activating NFAT and promoting their transfer into the nucleus, and interacting with interleukin-2 initiation factor AP-1-related gene binding on Therefore, NFAT is closely related to the Ca ²⁺ -CaN dependent signaling pathway.

The NFAT family has five members, NFAT1/p, NFAT2/c, NFAT3, NFAT4/x and NFAT5, the first four of which are regulated by calcineurin. A variety of lymphomas, including RS cells in Hodgkin's lymphoma as well as diffuse large B-cell lymphoma and Burkitt's lymphoma, have shown decreased expression levels of NFATc1. Retroviral insertion label experiments showed that NFATc1 and NFAT5 genes were non-random insertion sites, and these genes were considered to be involved in the process of tumorigenesis. Infection of NFATc2 and/or NFATc3-deficient mice with lymphoma SL3-3 virus will lead to accelerated T-cell tumorigenesis, and NFATc3-deficient mice have a higher mortality rate than wild-type mice. These data suggest that NFAT transcription factors are critical for balancing lymphocyte activation and elimination and avoiding lymphomagenesis due to defective lymphocyte elimination or altered activation levels. In addition, NF-AT3 is the most important downstream step of the CaN-dependent signal transduction pathway. After being dephosphorylated by CaN, NF-AT3 translocates into the nucleus and plays a role in signal transmission. After being phosphorylated, NF-AT3 can be translocated from the nucleus. Therefore, the phosphorylation of NF-AT3 is also one of the means to inhibit the CaN signaling pathway, and it may also become the target to prevent cardiac hypertrophy, thereby providing cardiac-specific inhibitory drugs and avoiding immunosuppression, etc. complication.

On the one hand, NFAT plays a key role in the process of gene transcription caused by immune response, and it regulates the adaptive immune response system. On the one hand, NFAT plays a key role in the activation, proliferation, and apoptosis of lymphocytes, and it promotes activation-induced T/B cell death (AICD). Tumor suppressor, balances the activation and elimination of lymphocytes, and avoids the occurrence of lymphoma caused by defects in the elimination of lymphocytes or changes in the threshold level of activation. On the other hand, in T cells, NFAT, as a downstream substrate of CaN, can mediate the regulation of gene expression by Ca2 ⁺ signaling. Such an underlying mechanism coupling cellular Ca2 ⁺ signaling to cardiac gene transcriptional activation may also exist in the myocardium. In cardiomyocytes cultured in vitro, it was found that the activation of CaN, GATA4 and NT-AT could increase the activation of B-type natriuretic polypeptide (BNP) promoter by 100 times (Molkentin JD, Lu JR, Antos CL , et al. Cell, 1998). And this Ca2 ⁺ -activated, CaN-dependent signaling pathway may play an important role in the occurrence and development of cardiac hypertrophy. Moreover, CaN, as a multifunctional signaling enzyme, participates in the regulation of various cell functions. In addition to its role in the immune system and myocardium, it also plays an important role in the growth and development of nerve cells and the regulation of synaptic plasticity (Guerini et al. 1997) ; In skeletal muscle, may participate in the regulation of muscle fiber type conversion (Chin et al.1998). Previous studies have shown that CaN may be involved in the regulation of Ca ²⁺ in vascular smooth muscle cells (VSMC). Therefore, the transcriptional activity of NFAT is of great significance to the regulation of CaN-dependent signaling pathways such as the occurrence and development of the immune system and cardiac hypertrophy.

NFAT is a major target of immunosuppressants such as cyclosporin A and FK-506. They mainly suppress NFAT in the nucleus of T cells by inhibiting calcineurin, so that their genes cannot be transcribed and translated, thereby inhibiting IL-2, 3, 4, 8, IFNγ and other cytokines and IL-2 receptors and Expression of transferrin and inhibition of IgE-mediated allergic reactions. These biological immunosuppressants are one of the important trends in future drug development and have broad application prospects. Some have been approved by the US FDA, but most are in the experimental stage. Anti-IL-8 monoclonal antibody has been used in psoriasis with certain curative effect. The application of IL-10/11 can inhibit the production of IL-2 and IFN-γ by Th1 cells, contribute to the balance of Th1/Th2, and is effective for psoriasis (Ellis CN, Barker NWN.Psoriasis.CurrProb Dermatol, 2000, 12: 45-50). However, most of these drugs can inhibit CaN in non-immune cells, with poor selectivity and high toxicity and side effects. Therefore, it is necessary to develop reagents that have no toxic effects and can directly target NFAT, which will help to develop new, safe and effective drugs with strong selectivity. Immunomodulators.

Contents of the invention

In view of the deficiencies in the prior art and existing drugs or reagents, the purpose of the present invention is to provide a human new gene NFIF1 that inhibits the activation of human nuclear factor NF-κB and NFAT, and has determined that its expression product has the ability to inhibit NF-κB and NFAT The function of activation, and the effect of inhibiting the activation of NF-κB and NFAT is very obvious and stable. In addition, high exogenous expression of NFIF in cells can obviously lead to apoptosis of some cells. Therefore, the application of the gene in the preparation of pharmaceutical compositions for preventing and treating inflammation, allergic diseases, autoimmune diseases and tumors, and in regulating the activation, proliferation and apoptosis of lymphocytes and other immune regulation drugs is provided.

Technical scheme of the present invention comprises the following contents:

1. The polypeptide (327 amino acids) whose amino acid sequence is shown in Sequence 2 in the Sequence Listing, or its conservative variant polypeptide, or its active fragment.

2. A gene encoding the polypeptide of item 1 above.

3. The gene of item 2 above is characterized by having the nucleotide sequence (NFIF1) shown in Sequence 1 in the Sequence Listing, or a fragment thereof, or a sequence with a homology of more than 95%, especially more than 99%.

4. An expression vector containing the gene or fragment thereof in item 2 or 3 above.

5. The method for preparing the polypeptide of the above item 1, comprising transforming the expression vector of the gene NFIF1 or its fragment in the above item 4 into a host cell, culturing, and isolating the polypeptide from the culture.

6. A genetically engineered host cell characterized in that it is a host cell transformed or transduced with the gene described in item 2 above, or a host cell transformed or transduced with the vector described in item 3 above.

7. The use of the polypeptide of item 1 or the gene of item 2 in the preparation of drugs for the prevention and/or treatment of diseases related to human nuclear factor NF-κB, or in the preparation of drugs for regulating the activation and/or proliferation of lymphocytes and/or Use in a drug or reagent for apoptosis.

8. The use according to item 7 above, wherein the disease is selected from inflammation, allergic disease, autoimmune disease or tumor.

9. A pharmaceutical composition comprising the polypeptide of item 1 above and a pharmaceutically acceptable carrier.

10. An antibody capable of specifically binding to the polypeptide of item 1 above.

11. An in vitro detection method for inflammatory diseases or tumors, characterized by using the antibody of the above item 10 to detect the presence or level of the polypeptide in a sample from a host.

The polypeptide of the present invention may be a recombinant polypeptide, a synthetic polypeptide, etc., preferably a recombinant polypeptide. The polypeptides of the present invention include conservative variant polypeptides, or active fragments thereof, which refer to polypeptides that retain the same biological function or activity as the polypeptide.

Nucleotide sequences or fragments of the present invention include their complementary strands. The present invention also relates to a vector comprising the polynucleotide of the present invention, a genetically engineered host cell produced with the vector of the present invention and a polypeptide product of the present invention produced by recombinant technology. The polynucleotide sequences of the invention can be included in any of a number of expression vectors for expressing polypeptides. Suitable vectors include, for example, bacterial plasmids, adenoviruses, adeno-associated viruses, and the like. Expression vectors can be prokaryotic or eukaryotic expression vectors. The vector containing the gene sequence of the present invention can be used to transform appropriate host cells so that the host expresses the polypeptide.

The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a prokaryotic cell, such as Escherichia coli.

The polypeptide of the present invention can be mass-produced by gene recombination technology.

The polypeptide of the present invention has the effect of inhibiting the activation of human nuclear factor NF-κB and NFAT. The activation of human nuclear factor NF-κB is related to various diseases such as inflammation, allergic diseases, autoimmune diseases and tumors, and the activation of NFAT is related to the activation of lymphocytes. Activation, proliferation and apoptosis. Therefore, the polypeptide of the present invention can be effectively used for the prevention or treatment of these diseases.

The polypeptides of the present invention can be used alone or in combination with suitable pharmaceutical carriers. Such compositions may comprise a therapeutically effective amount of a polypeptide and a pharmaceutically acceptable carrier or excipient, such carriers including but not limited to: saline, buffered saline, dextrose, water, glycol, ethanol, or mixtures thereof, which should be Appropriate mode of administration.

The pharmaceutical composition of the present invention can be administered in an appropriate manner, such as topically, intravenously, intraperitoneally, intramuscularly, subcutaneously and other routes. The amount administered to a patient depends on many factors, such as the mode of administration, the physical condition of the treater and the experience of the physician.

The polypeptides of the present invention can also be used by expressing these polypeptides in vivo. For example, the patient's cells can be genetically engineered in vitro with the gene NFIF1 encoding the polypeptide of the present invention, and then the engineered cells are provided to the patient so that the engineered cells can highly express the polypeptide in vivo, thereby achieving the purpose of treatment.

The polypeptides of the present invention, or conservative variant polypeptides or fragments, or cells expressing them can be used as antigens to produce antibodies, and are clinically used for the clinical diagnosis, treatment, and curative effect of inflammatory diseases, allergic diseases, autoimmune diseases, and tumors It can also be used to study the molecular mechanism of NFIF1 inhibiting NF-κB and NFAT activation. The antibody can be a monoclonal antibody or a polyclonal antibody, and also includes chimeric, single-chain and humanized antibodies and Fab fragments, or products of a Fab expression library. Antibodies to a polypeptide of the present invention can be obtained by injecting the polypeptide directly into an animal. Methods of making monoclonal and polyclonal antibodies are well known in the art and described in numerous literatures. The antibody can be used to detect the presence or level of a polypeptide of the invention, or to detect a gene. In inflammation or tumor diseases, by detecting the level or presence of the polypeptide, it can be used to diagnose these diseases.

The NFIF1 gene was expressed in all normal tissues, fetal tissues, and tumor tissues used in the experiment, indicating that it is an important NFKB and NFAT regulatory molecule of the human body; the expression level is different in different tissues, indicating that it is in different tissues. The degree to which functions are performed in organizations varies.

Description of drawings

Fig. 1 Schematic diagram of cloning cDNA of NFIF1 gene coding region by pGEM-T Easy vector. The triangle icon is where the cDNA fragment of the NFIF1 gene coding region is inserted.

Fig. 2 Agarose gel electrophoresis of NFIF1 gene PCR amplification products in human tissues. In the figure: A is 14 kinds of human normal tissues, which are: A0 blank control, A1 heart, A2 pancreas, A3 testis, A4 ovary, A5 prostate, A6 colon, A7 small intestine, A8 skeletal muscle, A9 thymus, A10 lymph node, A11 tonsil, A12 white blood cells; B is 6 kinds of human tumor tissues, which are: B0 blank control, B1 lung cancer, B2 pancreatic cancer, B3 ovarian cancer, B4 prostate cancer, B5 colon cancer, B6 breast cancer; C is 8 kinds of fetuses Tissues, which are: C0 blank control, C1 fetal lung, C2 fetal heart, C3 fetal liver, C4 fetal spleen, C5 fetal kidney, C6 fetal brain, C7 fetal skeletal muscle, C8 fetal thymus; M is the molecular weight marker; Figure 2 Figures 2-1 and 2-2 are the agarose gel electrophoresis patterns of the PCR amplification product of NFIF1 gene, and Fig. 2-3 is the agarose gel electrophoresis pattern of the PCR amplification product of the housekeeping gene G3PDH.

Fig. 3 Schematic diagram of the construction of the eukaryotic expression vector pcDNA3.1B-NFIF1.

Fig. 4 Structural diagram of pNF-KB-luc and pNFAT-luc luciferase gene reporter plasmids. The upper figure is the structural diagram of the pNF-κB-luc luciferase gene reporter plasmid, and the lower figure is the structural diagram of the pNFAT-luc luciferase gene reporter plasmid.

Figure 5 NFIF1 inhibits NF-κB activation and TNF-induced NF-κB activation. 293T cells were co-transfected with pcDNA3.1B and pNF-κB-luc, and the luciferase activity ratio (firefly luciferase activity/jellyfish luciferase activity) was set to 1 when there was no stimulation, and the left column was blank , and the right square column is TNFα.

Figure 6 NFIF1 inhibits NF-κB activation and PMA+ionomycin-induced NF-κB activation.

In the figure: 293T cells were co-transfected with pcDNA3.1B and pNF-κB-luc, and the luciferase activity ratio was set to 1 when there was no stimulation. The left square column was P+I, and the right square column was blank.

Figure 7 NFIF1 inhibits MEKK-induced NF-κB activation. The ratio of luciferase activity when pcDNA3.1B and pNF-κB-luc were co-transfected into 293T cells was set as 1. Among them, the left square column is TNFc, the middle square column is P+I, and the right square column is blank.

Figure 8 NFIF1 inhibits NFAT activation and PMA+ionomycin-induced NFAT activation. In the figure: 293T cells were co-transfected with pcDNA3.1B and pNFAT-luc, and the luciferase activity ratio was set as 1 when there was no stimulation. Among them, the left square column is P+I, and the right square column is blank. The data in Figs. 5-8 are the results obtained from three repeated experiments.

Embodiments of the present invention are further described in detail below.

1. Isolate the cDNA of NFIF1 gene from human tissue, determine its nucleotide sequence, and further deduce its coded amino acid sequence.

In the present invention, the human function unknown gene sequence (Ref: XM_114657; Locus ID: 203245; UniGene: Hs.373606) is obtained by searching the NCBI nr database. The nucleotide sequence is as sequence 3 in the sequence list shown.

Further use the Human_est database to perform sequence correction by the BLASTn method, and the corrected sites are as follows:

1) The 37th base is corrected from G to A;

2) Base 562 was corrected from T to C;

3) 45 bases were inserted between base 1283 and base 1284, and it was predicted that this insertion would not change the ORF (open reading frame);

4) Extend 405 bases after the last base of the sequence, and it is predicted that this extension will not change the ORF; the corrected sequence is shown as sequence 4 in the sequence listing.

In addition, there is an alternative splicing body analyzed, and it is predicted that this splicing body does not change the ORF, and its difference from the original sequence is located in the 3' untranslated region. The sequence is shown in sequence 5 in the sequence listing.

The gene was named NFIF1. The chromosome location of NFIF1 gene is 9q34.13, which consists of 3 exons and 2 introns (its spliced body consists of 2 exons and 1 intron). The predicted full-length ORF is 984bp, encoding 327 amino acids.

According to the NFIF1 gene sequence, design NFIF1 gene-specific primers (5' primer: 5'-gggggtgggtgaggggc-3'; 3' primer: 5'-acccctgccctcactggatg-3') from mixed human tissue cDNA library (brain, lung, colon, testis , Clometech, K14201-1, K1241-1) to obtain the cDNA fragment of the NFIF1 gene coding region by PCR amplification. TagDNA polymerase was used in the PCR amplification to obtain the amplified product containing the 3' protruding base A, which was recovered and purified after electrophoresis identification and inserted into the linear pGEM-T Easy vector containing the 3' protruding base T (Promega, A1360), transform Escherichia coli DH50α, extract the plasmid, and use ABI PRISM 3700 DNA analyzer (Perkin-Elmer/applied Biosystem) to sequence to obtain NFIF1 gene cDNA fragment 1091bp (wherein 984bp is the open reading frame region). The results of sequence determination confirmed that the 562 bases in the sequence correction were corrected from T to C, while the corrections of other positions were not confirmed because they were not within the scope of PCR amplification. The sequencing results are shown in sequence 1.

The schematic diagram of the PCR amplified fragment of NFIF1 gene coding region cDNA cloned by pGEM-T Easy vector is shown in Figure 1.

According to the sequencing results, the amino acid sequence of the protein encoded by the NFIF1 gene is shown in Sequence 2.

2. Construction of eukaryotic expression vector

In order to detect the inhibitory function of NFIF1 on the activation of NF-κB and NFAT, the eukaryotic expression vector pcDNA3.1B-NFIF1 (hereinafter abbreviated as pcDB-NFIF1) containing NFIF1 cDNA was constructed first. The present invention adopts eukaryotic expression vector pcDNA3.1/MycHis(-)B (Invitrogen, V85520, hereinafter abbreviated as pcDB) and NFIF1 gene recombination to construct eukaryotic expression vector. pcDB is a vector designed for high expression of recombinant proteins in mammalian cell lines. It contains the human cytomegalovirus CMV promoter, which can achieve high expression in mammalian animal cell lines. The NFIF1 cDNA inserted into the pGEM-T Easy vector was excised with EcoRI, and pcDB was digested with EcoRI at the same time. After the two were connected, they were transformed into Escherichia coli DH5α, and the transformants were selected to extract the plasmid. The correct forward insertion clone was selected by sequencing, and the clone containing The eukaryotic expression vector pcDB-NFIF1 of NFIF1 cDNA, Figure 3 is a schematic diagram of constructing the eukaryotic expression vector.

3. The method for detecting the function of NFIF1 gene inhibiting the activation of NF-κB and NFAT

Human embryonic kidney 293T cells (ATCC Number: CRL-11268) were co-transfected with pcDB-NFIF1 and pNF-κB-luc (Stratagane, 219077), pRL-SV40 (Promega, E2231), using the dual luciferase reporter gene method, The effects of the expression products of NFIF1 cDNA in 293T cells on the activation of NF-κB induced by TNFα, PMA (Sigma) + ionomycin (Calbiochem) and MEKK and the activation of NFAT induced by PMA (Sigma) + ionomycin (Calbiochem) were determined. inhibition.

The present invention adopts the dual luciferase reporter gene method to detect the inhibitory function of NFIF1 gene on NF-κB activation. The method adopts the in vivo signal transduction pathway cis reporter system, wherein the pNF-κB-luc reporter plasmid used is loaded with firefly Luciferase gene, whose expression is regulated by a synthetic promoter, which contains the basic promoter element (TATA box), and the binding site of the transcription factor NF-κB, the structure of the pNF-κB-luc reporter plasmid As shown in Figure 4. When NF-κB in the cell is activated through a certain signal transduction pathway, NF-κB will bind to the enhancer element of the reporter plasmid pNF-κB-luc, thereby initiating the transcription of the reporter gene firefly luciferase gene, Further intracellular translation into luciferase leads to an increase in the number and activity of luciferase in the cell; on the contrary, when the activation of NF-κB in the cell is inhibited, the expression and activity of luciferase are low. Therefore, by detecting the activity of luciferase, it can reflect whether different stimuli and co-transfected target genes have the function of activating or inhibiting the activation of intracellular NF-κB. The principle of use of the pNFAT-luc reporter plasmid is similar to that of the pNF-κB-luc reporter plasmid. The pRL-SV40 reporter plasmid contains the jellyfish luciferase gene, whose expression is regulated by the Simian virus 40 (SV40) enhancer/promoter, and can produce strong basal levels of jellyfish luciferase after transfection into mammalian cells The expression of , and the expressed activity is not regulated by the activation of NF-κB and NFAT, so it is used as an internal control reporter gene in the experiment.

Plasmids pcDB-NFIF1 and pNF-κB-luc, pRL-SV40 were co-transfected into 293T cells. 24 hours after transfection, the 293T cells were activated with TNFα or PMA+ionomycin. After 8 hours, the cells were lysed and the activity of luciferase was detected. . The measured results showed that the expression product of NFIF1 gene in 293T cells could inhibit the activation of NF-κB. It shows that NFIF1 has a negative regulatory effect on NF-κB.

The method for detecting the function of NFIF1 gene to inhibit the activation of NFAT is the same as above, only PMA+ionomycin is used to activate 293T cells. The measured results showed that the expression product of NFIF1 gene in 293T cells could inhibit the activation of NFAT. It shows that NFIF1 has a negative regulatory effect on NFAT.

Advantages of the present invention:

1. Provided the cDNA sequence of the new human gene NFIF1 and its encoded polypeptide, and found for the first time that the transfection of this gene has the function of inhibiting the activation of NF-κB;

2. NFIF1 has the effect of simultaneously inhibiting the activation of NF-κB and NFAT, and is highly efficient and stable, indicating that NFIF1 has relatively high affinity and efficacy in cells;

3. NFIF1 is expressed in most normal cells in the body, indicating that it is an important regulator of NFκB and NFAT.

4. Based on the above three advantages, the present invention aims to further study the regulatory relationship between NFIF1, NF-κB and NFAT, and develop and treat inflammation, allergic diseases, autoimmune diseases and tumors, and regulate the activation, proliferation and activation of lymphocytes. The new drugs for apoptosis will lay the necessary foundation for the development of new clinical diagnosis, curative effect evaluation and prognostic indicators.

specific implementation plan

Cloning of embodiment 1, NFIF1 gene cDNA

Searched the predicted genes with unknown human functions in the nr database of NCBI, and obtained the sequence of human unknown functional genes (Ref: XM_114657; Locus ID: 203245; UniGene: Hs.373606), which was set as sequence 3, and was obtained by BLASTn using the Human_est database. Methods Sequence correction was performed, and the final sequence was set as sequence 4. Design NFIF1 gene-specific primers according to sequence sequence 4:

5' Primer: 5'-GGGGGTGGGTGAGGGG-3'

3' Primer: 3'-ACCCCTGCCCTCACTGGATG-3'

Use the above primers to carry out PCR amplification reaction with mixed human tissue cDNA library (brain, lung, pancreas, testis, Clonetech K1420-1, K1241-1) as template, and the reaction conditions are as follows:

The reaction volume is 50 μl, which contains:

Mixed human tissue cDNA template 2μl (0.5μl each for brain, lung, colon, and testis)

The final concentrations of primers 5' primers and 3' primers are 0.2 μM each

dNTP final concentration 200μM each

Taq DNA Polymerase 2.5U

10×Taq DNA polymerase buffer 5μl

Make up to 50 μl volume with double distilled water.

Reaction temperature and time: 94°C, after denaturation for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 63°C for 30 seconds, extension at 72°C for 80 seconds, 30 cycles of amplification, and finally extension at 72°C for 7 minutes.

The amplified product is a 3' protruding sticky-end fragment with a base A at the 3', purified with the QIA Quick Gel Extraction Kit (Qiagen, 28704) according to the product instructions, and then combined with a linear pGEM-T EASY with a base T at the 3' Carrier (Promega, A1360) was ligated overnight at 16°C, using 2mm electrode cup, 2500V to transform Escherichia coli DH5α, the transformant was grown on LB plate medium containing ampicillin, clones were selected, plasmids were extracted, and AbI PRISM 3700 DNA analyzer was used (Perkin-Elmer/Applied Biosystem) sequencing to obtain cDNA1091bp of NFIF1 gene, in which ORF is 984bp in full length and encodes 327 amino acids.

Embodiment 2, RT-PCR method detects the transcription product of NFIF1 gene in human tissue with the specific primer of NFIF1 gene:

5' Primer: 5'-TGTGTCCGTCGCCATGACAG-3'

3' Primer: 5'-TGGTTGAGTGGCAGGTGAGG-3'

12 kinds of human normal tissues (heart, pancreas, testis, ovary, prostate, colon, small intestine, skeletal muscle, thymus, lymph nodes, tonsils, white blood cells); 6 kinds of human tumor tissues (lung cancer, pancreatic cancer, ovarian cancer, prostate cancer) , colon cancer, breast cancer); and cDNA libraries of 8 kinds of fetal components (fetal lung, fetal heart, fetal liver, fetal spleen, fetal kidney, fetal brain, fetal skeletal muscle, fetal thymus) (Clonetch, K1420-1, 1241-1) was used as template for PCR amplification.

The PCR amplification conditions are as follows:

The reaction volume is 50 μl, which contains:

Mixed human tissue cDNA template 2μl (0.5μl each for brain, lung, colon, and testis)

The final concentrations of primers 5' primers and 3' primers are 0.2 μM each

dNTP final concentration 200μM each

Taq DNA Polymerase 2.5U

10×Taq DNA polymerase buffer 5μl

Make up to 50 μl volume with double distilled water.

Reaction conditions:

After denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, amplification for 30 cycles, and finally extension at 72°C for 7 minutes. In addition, housekeeping gene-specific primers: 5' primer: 5'-ACCACAGTCCATGCCATCAC-3', 3' primer: 5'-TCCACCACCCTGTTGCTGTA-3' were used for PCR amplification with the same template as above, respectively, as an internal control. The PCR reaction system is the same as before, and the reaction conditions are as follows: 94°C, denaturation for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, 25 cycles of amplification, and finally extension at 72°C for 7 minutes . PCR reaction result is as shown in Figure 2, and the result of amplification shows, all exists the cDNA of NFIF1 gene in the cells of these tissues (referring to Fig. 2), illustrates that NFIF1 has all produced the transcription product of NFIF1 gene in the cells of these tissues, has Broad expression profile, involved in the regulation of transcription factors in a variety of tissues.

Embodiment 3, the construction of NFIF1 gene eukaryotic expression vector

The cDNA fragment of NFIF1 gene was excised from the pGEM-T Easy vector (Promega, A1360) with EcoRI (Promega), and the eukaryotic expression vector pcDNA3.1/mycHis(-)B (Invitrogen, V85520) was digested with EcoRI simultaneously, According to the method described in J.Sambrook et al., Molecular Cloning Experiment Guide 2nd Edition, the digested NFIF1 was connected to the carrier overnight at 16°C, transformed into Escherichia coli DH5α, and the transformant was grown on LB plate medium containing ampicillin , select the growing colony, extract the plasmid, digest with EcoRI, identify the digested product by agarose gel electrophoresis, select the positive clone with the insert, and select the correct positive clone by sequencing (using ABI PRISM3700 DNA analyzer, as above). The insert clone was named pcDNA3.1B-NFIF1.

At the same time, the culture solution was collected, and the precipitated protein was analyzed by SDS-PAGE to obtain the NFIF1 polypeptide.

The analysis results of NFIF1 protein showed that the protein sequence of NFIF1 is shown in Sequence 2 of the Sequence Listing, with a molecular weight of 35.1 kD and an isoelectric point of 6.82. Transmembrane region analysis (TMHMM analysis, http://www.cbs.dtu.dk/services/TMHMM) showed no transmembrane region. Nuclear localization signal analysis showed no obvious nuclear localization signal. Analysis of subcellular localization suggested that the possibility of being located in the nucleus was the highest (nucleus: 39.1%; mitochondria: 30.4%; cytoplasm: 17.4%; secretion system vesicles: 8.7%; cytoskeleton: 4.3).

Example 4. Determination of the inhibitory effect of NFIF1 on the activation of NF-κB induced by TNFα, PMA+ionomycin by dual luciferase reporter gene method.

Human embryonic kidney 293T cells were co-transfected with the target gene pcDB-NFIF1 and the reporter genes pNF-κB-luc and pRL-SV40, and the activity of NF-κB was determined by detecting the two luciferase activities respectively. The method of co-transfection was liposome transfection method, using LipfectanineTM2000 (Invitrogen, 11668027), as described in the product manual.

The transfection steps are as follows:

(1) Cell culture: use 293T cells (ATCC Number: CRL-11268) (2.0×104) with DMEM (Dulbecco's modified Eagle's medium) medium (Hyclone, SH0022.02) containing 10% fetal bovine serum Spread on 96-well cell culture plate (Costar, 3599) and culture in 5% CO2, 37°C incubator for 16 hours.

(2) Preparation of DNA-LipfectamineTM2000 complex: Dilute 20ng pNF-KB-luc, 2ngpRL-SV40 and 0-80ngpcDB-NFIF1 with 25μl serum-free DMEM medium, mix slowly and evenly; dilute appropriate amount with 25μl DMEM medium LipfectaminTM2000, slowly mixed evenly, incubated at room temperature for 5 minutes, slowly mixed with diluted DNA, and left at room temperature for 20 minutes to form DNA-Lipofectamine 2000 complex.

(3) Transfection: slowly drop the DNA-Lipofectamine 2000 complex into the cell culture plate (50 μl/well), and shake slightly. 5% CO2, 37 ° C incubator for 24 hours.

After 24 hours, TNFα (tumor necrosis factor α, IYX300-01A2, final concentration 20ng/ml) or PMA (phorbol myristate, Calbiochem, 524400, final concentration 50ng/ml) + ionomycin (Calbiochem, 407950, Final concentration 1nM) to stimulate cells, or no stimulant; Discard the medium after 8 hours, add Reporter Lysis Buffer (Promega, E4030), place at -80°C for 30 minutes, take it out and let it thaw naturally at room temperature to lyse the cells , and then the cell lysate was pipetted into a fluorescent plate (Gronier, 655075), and the luciferase activity was detected by Fluostar OPTIMA (BMGLabtechnologies) with Dual-Luciferase Reporter Assay System 10-Pack (Promaga, E1960).

Set the intracellular luciferase activity ratio (firefly luciferase fluorescence intensity/jellyfish luciferase fluorescence intensity) to 1 when the pcDB empty vector is transfected and no stimulus is added, and the intracellular luciferase activity under other conditions The ratio is based on this as a standard and expressed as a relative value.

result:

(1) Inhibitory effect of NFIF1 on TNFα-induced NF-κB activation

Referring to Figure 5, 293T cells were co-transfected with 0, 10, 20, 40, 80ng pcDNA3.1B-NFIF1 and 20ngpNF-B-luc, respectively, and stimulated with TNFα as the amount of pcDNA3.1B-NFIF1 in co-transfection increased. The activity of luciferase in 293T cells decreased obviously rapidly, while the activity of luciferase in 293T cells not stimulated with TNFα decreased slowly, indicating that the expression product of NFIF1 in 293T cells has a strong effect on the activation of NF-κB induced by TNFα. inhibitory effect.

(2) Inhibitory effect of NFIF1 on PMA+ionomycin-induced NF-κB activation

Referring to Figure 6, 293T cells were co-transfected with 0, 10, 20, 40, 80ng pcDNA3.1B-NFIF1 and 20ng pNF-κB-luc, respectively. With the increase of dosage, the activity of luciferase in 293T cells stimulated with PMA+ionomycin significantly decreased rapidly, while the activity of luciferase in 293T cells not stimulated with PMA+ionomycin decreased slowly, which also indicated that NFIF1 was activated in 293T cells. The expression product in cells has a strong inhibitory effect on the activation of NF-κB induced by PMA+ionomycin.

Example 5. Dual-luciferase reporter gene method was used to determine the inhibitory effect of NFIF1 on MEKK-induced NF-κB activation. In addition to co-transfecting 293T cells with 20ng pNF-κB-luc, 2ngpRL-SV40, 0-80ngpcDNA3.1B-NFIF1 (complemented with pcDNA3.1B vector when less than 80ng) and 20ngpFC-MEKK (Stratageme, 219077), and transfecting 24 After 1 hour, the cells were directly lysed, and the rest were the same as in Example 4.

result:

Referring to Figure 7, 293T cells were co-transformed with 0, 10, 20, 40, 80ng pcDNA3.1B-NFIF1 and 20ngpNF-κB-luc, 2ngpRL-SV40 and 20ng pFC-MEKK, respectively. The inhibitory effect was similar to that induced by TNF, PMA+ionomycin, thus indicating that NFIF1 has a strong inhibitory effect on MEKK-induced NF-κB activation.

According to the above experimental results, it can be determined that when the activation of NF-κB causes the transcriptional expression activation of some disease-related genes, the inhibition of NF-κB activation by NFIF1 can prevent the occurrence and development of the disease. Therefore, the present invention can be used to prepare medicines for preventing and treating inflammation, allergic diseases, autoimmune diseases and tumors.

Example 6. The dual luciferase reporter gene method was used to determine the inhibitory effect of NFIF1 on the activation of NFAT induced by PMA+ionomycin.

Except that 20ng pNFAT-luc was used instead of pNF-κB-luc for transfection, the other experiments were the same as in Example 4 for the inhibitory effect of NFIF1 on PMA+ionomycin-induced NF-κB activation.

result:

Referring to Figure 8, 293T cells were co-transfected with 0, 10, 20, 40, 80ng pcDNA3.1B-NFIF1 and 20ngPnfat-luc respectively. The activity of luciferase in the 293T cells stimulated by PMA + ionomycin significantly decreased rapidly, while the activity of luciferase in the 293T cells stimulated by PMA + ionomycin decreased slowly, which also showed that the expression product of NFIF1 in 293T cells had a strong effect on PMA + ions. Mycin-induced NFAT activation has a strong inhibitory effect.

Therefore, the present invention can be used in the development of new drugs that regulate the activation, proliferation and apoptosis of lymphocytes.

Embodiment 7, antibody preparation

The antigen is selected from the full-length or partial peptides of NFIF1 protein expressed by prokaryotic cells or eukaryotic cells, and a synthetic polypeptide can also be used as the antigen.

Preparation of polyclonal antibody: select adult male New Zealand rabbits or BALb/c mice as immunized animals, and use 200ug (New Zealand rabbits) or 20ug (BALb/c mice) antigen and an equal volume of complete Freund's adjuvant (FCA) for the first immunization After emulsification, inject multiple points subcutaneously on the back. 21, 42, and 63 days after the initial immunization, the antigen protein fully emulsified with Freund's incomplete adjuvant (FIA) was boosted once each, and the dosage was the same as before. 7-10 days after each immunization, the serum titer was detected by ELISA method, and when it reached 1×10-4, the serum was separated by bloodletting. Western blot was used to identify the specificity of the antibody.

Preparation of monoclonal antibody: Immunize BALb/c mice as before, take spleen to make B cell suspension, fuse with myeloma cells SP2/0 in logarithmic growth phase, pass HAT (H, hypoxanthine; A, aminopterin T, thymidine) was selectively cultured to obtain hybrid cell lines, and then the antibody titer was detected by ELISA method to screen specific hybridoma cell lines and obtain monoclonal antibodies.

NFIF1 antibody can be used clinically for inflammatory diseases, allergic diseases, autoimmune diseases and tumor clinical diagnosis, treatment, efficacy evaluation, etc., and can also be used for research on the molecular mechanism of NFIF1 inhibition of NF-κB and NFAT activation.

SEQUENCE LISTING

<110>Peking University Beijing Nuosai Genome Research Center Co., Ltd.

<120> A gene that inhibits the activation of human nuclear factor NF-κB and NFAT, its encoded polypeptide and its application

<130>

<160>5

<170>PatentIn version 3.1

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                 Met Ala Val Pro Ala Lys

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aaa agg aag atg aac ttc tca gag cgg gag gtg gag atc atc gtg gag 163

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Glu Leu Glu Leu Lys Lys His Leu Leu Val Asn His Phe Asn Ala Gly

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Val Pro Leu Ala Ala Lys Ser Ala Ala Trp His Gly Ile Leu Arg Arg

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Val Asn Ala Val Ala Thr Cys Arg Arg Glu Leu Pro Glu Val Lys Lys

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Arg Ala Ala Val Glu Gly Gly Glu Ala Pro Gly Pro Thr Glu Glu Asp

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Gly Ala Gly Gly Pro Gly Thr Gly Gly Gly Ser Gly Gly Gly Gly Pro

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Ala Val Ala Pro Val Leu Leu Thr Pro Met Gln Gln Arg Ile Cys Asn

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Leu Leu Gly Glu Ala Thr Ile Ile Ser Leu Pro Ser Thr Thr Glu Ile

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His Pro Val Ala Leu Gly Pro Ser Ala Thr Ala Ala Ala Ala Thr Val

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acc ctg aca cag atc ccc aca gag acc acc tat cac acg ctg gag gag 643

Thr Leu Thr Gln Ile Pro Thr Glu Thr Thr Tyr His Thr Leu Glu Glu

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Gly Val Val Glu Tyr Cys Thr Ala Glu Ala Pro Pro Pro Leu Pro Pro

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Glu Thr Pro Val Asp Met Met Ala Gln His Ala Asp Thr Ser Val Lys

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ccg caa gcg ctc aag agc cgc att gct ctc aac tcc gcc aag ctg ata 787

Pro Gln Ala Leu Lys Ser Arg Ile Ala Leu Asn Ser Ala Lys Leu Ile

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Val Gln Ala Cys Ala Gln Glu Arg Gln Ala Gln Ala Met Glu Gly Thr

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Asp Phe Arg Arg Tyr Let Gln Ser Asn Thr Ala Asn Pro Ala Pro Ala

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Ser Asp Pro Gly Gln Val Ala Gln Asn Gly Gln Pro Asp Ser Ile Ile

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Val Glu Ile Ile Val Glu Glu Leu Glu Leu Lys Lys His Leu Leu Val

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Asn His Phe Asn Ala Gly Val Pro Leu Ala Ala Lys Ser Ala Ala Trp

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His Gly Ile Leu Arg Arg Val Asn Ala Val Ala Thr Cys Arg Arg Glu

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Leu Pro Glu Val Lys Lys Lys Trp Ser Asp Leu Lys Thr Glu Val Arg

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Arg Lys Val Ala Gln Val Arg Ala Ala Val Glu Gly Gly Glu Ala Pro

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Gly Pro Thr Glu Glu Asp Gly Ala Gly Gly Pro Gly Thr Gly Gly Gly

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Ser Gly Gly Gly Gly Pro Ala Val Ala Pro Val Leu Leu Thr Pro Met

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Gln Gln Arg Ile Cys Asn Leu Leu Gly Glu Ala Thr Ile Ile Ser Leu

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Pro Ser Thr Thr Glu Ile His Pro Val Ala Leu Gly Pro Ser Ala Thr

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Ala Ala Ala Ala Thr Val Thr Leu Thr Gln Ile Pro Thr Glu Thr Thr

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Tyr His Thr Leu Glu Glu Gly Val Val Glu Tyr Cys Thr Ala Glu Ala

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Ala Asp Thr Ser Val Lys Pro Gln Ala Leu Lys Ser Arg Ile Ala Leu

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Lys Glu Ile Ala Gln His Leu Glu Gln Gln Asn Asp Leu Leu Gln Met

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Ile Arg Arg Ser Gln Glu Val Gln Ala Cys Ala Gln Glu Arg Gln Ala

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Gln Ala Met Glu Gly Thr Gln Ala Ala Leu Ser Val Leu Ile Gln Val

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ggtcaagaag aagtggtctg acctcaagac cgaggtccgt cgcaaggttg cccaggtccg 480

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cacagagatc caccctgtgg ccctcggacc ctcggccacc gcagccgcag ccacggtcac 720

cctgacacag atccccacag agaccaccta tcacacgctg gaggagggcg tggtggagta 780

ctgcacggct gaggcgcccc cacctctgcc accagagacc cctgtggaca tgatggccca 840

gcatgcagac acgtcggtca agccgcaagc gctcaagagc cgcattgctc tcaactccgc 900

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ggaacagcag aacgacctac tgcagatgat ccgccgctcc caggaagtgc aggcctgtgc 1020

ccaggagcgc caggcccagg ccatggaggg cacacaggct gccctgagcg tcctcatcca 1080

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tcaccatcaa ttaccaaggc ccttgcatct ccccagatcg tttcccaagt ttggcgattt 1320

gctgtcggtg cctcagccac atatgaccaa tggttacaac tcagggctcc agacctcagc 1380

taaaaagaga agacgctgcc ctcctgggca cgaacgttta gaatgctcaa ctcctctatt 1440

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aaaaaa 1866

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ctggcacggc atcctgagaa gggtcaacgc cgtggccacc tgccgcagag agctgcctga 420

ggtcaagaag aagtggtctg acctcaagac cgaggtccgt cgcaaggttg cccaggtccg 480

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cctgacacag atccccacag agaccaccta tcacacgctg gaggagggcg tggtggagta 780

ctgcacggct gaggcgcccc cacctctgcc accagagacc cctgtggaca tgatggccca 840

gcatgcagac acgtcggtca agccgcaagc gctcaagagc cgcattgctc tcaactccgc 900

caagctgata caggagcagc gggtcaccaa cctgcatgtg aaggagatcg cacagcacct 960

ggaacagcag aacgacctac tgcagatgat ccgccgctcc caggaagtgc aggcctgtgc 1020

ccaggagcgc caggcccagg ccatggaggg cacacaggct gccctgagcg tcctcatcca 1080

ggtcctccgg cctatgatca aagatttccg ccgctacctg cagagcaaca cagctaaccc 1140

ggcccccgcc tctgaccctg ggcaggtggc ccagaatggg cagccagaca gcatcatcca 1200

gtgagggcag gggtcaggcc agccttctgc catgatggga tgaaaactcc atggacttgt 1260

aagtgggtcc tgtgattggc cttggcctta gaccggccac gtgcacagct ccctctttaa 1320

taaacgctta ggggttgcac tgttaaaaaa aaaaaaaaaa aa 1362

Claims (11)

1. A polypeptide whose amino acid sequence is shown in Sequence 2 in the Sequence Listing, or a conservative variant polypeptide thereof, or an active fragment thereof. 2. A gene encoding the polypeptide of claim 1. 3. The gene of claim 2, characterized by having the nucleotide sequence (NFIF1) shown in Sequence 1 in the Sequence Listing, or a fragment thereof, or a sequence with more than 95% homology. 4. An expression vector comprising the gene of claim 2 or 3 or a fragment thereof. 5. The method for preparing the polypeptide according to claim 1, comprising transforming the expression vector of the gene or its fragment according to claim 4 into a host cell, culturing, and isolating said polypeptide from the culture. 6. A genetically engineered host cell, characterized in that it is a host cell transformed or transduced with the gene of claim 2, or a host cell transformed or transduced with the vector of claim 3. 7. Use of the polypeptide of claim 1 or the gene of claim 2 in the preparation of drugs for the prevention and/or treatment of diseases related to human nuclear factor NF-κB, or in the preparation of drugs for regulating the activation and/or proliferation of lymphocytes and/or or use in apoptotic drugs or reagents. 8. Use according to claim 7, wherein the disease is selected from inflammation, allergic disease, autoimmune disease or tumor. 9. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier. 10. An antibody capable of specifically binding to the polypeptide of claim 1. 11. An in vitro detection method for inflammatory diseases or tumors, characterized by using the antibody of claim 10 to detect the presence or level of the polypeptide in a sample from a host.

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