CN100368436C - Molecular design and preparation method of human chorionic gonadotropin recombinant multi-epitope chimeric peptide CP22 antigen - Google Patents

Abstract

The present invention provides human chorionic gonadotropin recombination multi-epitope gomphosis peptide CP22, which is basically composed of epitope peptide HBsAg<19-33>, epitope peptide HBcAg <85-140>, epitope peptide TT5 <80-599>, and two groups of beta5 epitope peptide, beta8 epitope peptide and beta9 epitope peptide of human chorionic gonadotropin. The present invention also provides a nucleotide sequence for encoding the peptide, an expression carrier with the sequence, a host cell, and a method for preparing the gomphosis peptide; the gomphosis peptide of the present invention can be used for preparing the vaccine composition for treating tumors and / or contraception.

Description

Molecular design and preparation method of human chorionic gonadotropin recombinant multi-epitope chimeric peptide CP22 antigen

technical field

The invention relates to the technical field of genetic engineering, more specifically, the invention relates to human chorionic gonadotropin recombinant multi-epitope chimeric peptide CP22 antigen and a preparation method thereof.

Background technique

Human chorionic gonadotropin (hCG), also known as pregnancy hormone, is a glycoprotein hormone that maintains early pregnancy in humans. It consists of two subunits, α and β. In addition, it has also been identified as a carcinoembryonic antigen, which is expressed in many malignant tumors, for example, hCG can be detected in cancers of the pancreas, lung, colorectum, cervix, ovary, liver, stomach, etc. and the existence of its subunits or fragments (Triozzi P and StevensVC. Human chorionic gonadotropin as a target for cancer vaccines. Oncol Rep 6: 7-17, 1999). Therefore, hCG has been considered as an important target antigen for the development of contraceptive and/or tumor therapeutic vaccines. As a vaccine, the hCGβ subunit purified and split from natural hCG and the hCGβ carboxyl-terminal (carboxyl-terminal peptide, CTP) containing β9 and β8 two specific B-cell epitopes to synthesize 37 peptide antigens are the most widely used so far. . Although the hCGβ heterodimer (HSD-hCG) vaccine coupled with the sheep LHα subunit has passed the human clinical phase II efficacy test (Talwar GP, et al.A vaccine that prevents pregnancy in women.Proc Natl Acad SciUSA, 1994 ; 91:8523-8536), verified the feasibility of immunocontraception and has a milestone significance, but it is not suitable for promotion and application due to some insurmountable defects and practical problems (Stevens VC.Progress in the development of human chorionic gonadotropin antifertility vaccines .Am J ReprodImmunol, 1996;35:148-155.Talwar GP, Singh Om, Gupta SE, et al.The HSD-hCGvaccine prevents pregnancy in women: feasibility study of a reversible safe contraceptive vaccine.Am J Reprod 97Immunol, 71 : 153-160). For example: the purification and resolution of hCGβ antigen is not easy and costly; the vaccinogen needs to be coupled with sheep LH α subunit and tetanus toxoid (TT) or diphtheria toxoid (DT), and because it is a self-antigen with weak immunogenicity. Special adjuvants, in addition to greatly increasing the cost of vaccines, are also difficult to mass-produce vaccines according to GMP standards; in human clinical phase II efficacy trials, 20% of women tested do not respond to vaccines or respond ineffectively. The hCGβ-CTP synthetic peptide vaccine also has similar problems, and the corresponding human clinical phase II efficacy trials have not been successfully implemented. However, it has recently passed human clinical phase II efficacy trials as a therapeutic vaccine for hCG-dependent malignancies (Moulton HM, et al. Active specific immunotherapy with aβ-human chorionic gonadotropin peptide vaccine in patients with metastatic colorectal cancer: antibody response is associated with improved survival. Clin Cancer Res, 2002; 8(7): 2044-2051), the effect of active immunosuppression of tumor growth or prolonging the survival of patients is obvious, thus demonstrating the application of hCGβ-CTP synthetic peptide vaccine in tumor immunotherapy prospect. But needless to say, the actual human clinical application also needs to overcome the problem of coupling with DT, the need for special adjuvants (to reduce the cost of the vaccine and facilitate the production of preparations), the specificity of the vaccine but the poor neutralization performance of the produced antibody against hCG, and how to improve And improve the immune response rate and treatment efficacy of tumor patients with different genetic backgrounds after vaccination.

On the other hand, the development of chemically synthesized peptide vaccines has always been expected to contribute to the prevention and treatment of human diseases. However, in the past forty years, a synthetic peptide vaccine that can be practically used has not been developed, although it has many such as Obvious advantages such as constant and relatively inexpensive rare antigens can be obtained. The main reason for this situation is that the target antigen single B-cell epitope (B-cell epitope, BCE) peptide is not enough to produce a complete immune effect. Nevertheless, those studies are still for the development of multi-epitope biosynthesis (also called recombinant) chimeric peptide vaccine trend (Xu WX.Trends in the development of chimeric peptide vaccines containing B-and T-cell epitopes.US Chin J Microbiol Immunol, 2000; 2(4):95-100) laid the groundwork, some of which suggested the incorporation of strong and/or "broad-spectrum" T-cell epitopes (T-cell epitopes) in synthetic peptides colinearly linked , TCE; Sinigaglia F, et al.A malaria T cell epitope recognized in association with most mouse and human class II molecules.Nature 336:778-780,1998; Panina-Bordignon P, etal.Universally immunogenic T cell epitopes: promis human class II and promiscuous recognition by T cells. Eur J Immunol, 1989; 19: 2237-2242), can act as a molecular adjuvant, and enable the target chimeric peptide vaccine to have the ability to overcome the genetic limitation of MHC immune response (Greenstein JL, et al. A universal T cell epitope-containing peptide from hepatitis B surface antigen canenhance antibody specific for HIV gp 120. J Immunol, 1992; 148: 3970-3977. Lou YH, et al. A zona ant pellucida le neptides vacc infertility without ovarian pathology. J Immunol, 1995; 155: 2715-2720), thus providing new ideas and new ways to simultaneously solve the problem of improving the immune response rate in people with different genetic backgrounds without special adjuvants . Limited by the length of chemically synthesized peptides, it is an inevitable choice to develop recombinant (also known as genetic engineering or biosynthesis) multi-epitope chimeric peptides (CP). There are few reports that peptide molecules can be expressed by a biological system and complete animal immunology studies, but only a few cases have shown the feasibility and development prospects of recombinant multi-epitope synthetic peptide vaccines, such as the combination expressed in Escherichia coli The CP of three glycoproteins and three B-cell epitopes of cap monkey ZP improved the immune function (Sivapurapu N, et al. Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro humansperm egg binding.Mol Reprod Dev, 2003;65:309-317), especially the combination of 12 B-cell epitopes and 6 helper T-cell epitopes and 1 broad-spectrum T-cell epitope expressed in insect cells Epitope Plasmodium CP immunogens not only generate strong specific antibody responses without special adjuvants, but also show the ability to overcome MHC restrictions (Shi YP, Hasnain SE, Sacci JB, et al. Immunogenicity and in In vitroprotective efficacy of a recombinant multistage Plasmodium falciparum candidate vaccine. Proc Natl Acad Sci USA, 1999; 96: 1615-1620. Shi YP, Das P, Holloway B, et al. Development, expression, and murine testing of a age multistage Plasma mod- eration falciparum malaria vaccine candidate. Vaccine, 2000, 18: 2902-2914).

In addition to the above hCG vaccines, especially the development status of hCGβ-CTP synthetic peptide vaccines and the enlightenment of new progress in the field of chemically synthesized peptides, the necessity of developing recombinant hCG multi-epitope CP is also based on the following report, that is, compounding and using hCGβ cyclic peptide ^38-57 (containing β5 linear B-cell epitopes ^45-52 ) and hCGβ-CTP ^109-145 (containing β9 and β8 specific linear B-cell epitopes ^{113-116, 137-144} ) two synthetic peptide vaccine experiments It is suggested that because the antibody response level of the 1:1 mixture of hCGβ cyclic peptide and hCGβ-CTP synthetic peptide (both coupled with DT) vaccine is significantly higher than the effect of the two immunizations alone, and compared with the antiserum of a single synthetic peptide vaccine, The ability of the antiserum produced by the mixture immunization to neutralize and bind target hCG was also significantly improved (Stevens VC. Am J Reprod Immunol, 1996; 35:148-155).

Contents of the invention

One aspect of the present invention provides an isolated polypeptide, which is human chorionic gonadotropin recombinant multi-epitope chimeric peptide CP22, which basically consists of epitope peptide HBsAg ^19-33 , epitope peptide HBcAg ^85-140 and Epitope peptide TT ^580-599 , and two groups of human chorionic gonadotropin β5 epitope peptide, β8 epitope peptide and β9 epitope peptide.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state. The term "isolated polypeptide" means that the polypeptide of the present invention is substantially free of other proteins, lipids, carbohydrates or other substances. Those skilled in the art can purify the polypeptide using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The term "consisting essentially of" means that the polypeptide may also contain any other amino acids, as long as the presence of these amino acids has no substantial adverse effect on the biological effect of the chimeric peptide of the present invention. As shown below, there may also be amino acid fragments that enhance the hydrophilicity of the chimeric peptides or flexible linkers or corner peptides that improve the molecular structure of the protein between each epitope peptide in the chimeric peptide of the present invention.

The order of each epitope peptide contained in the chimeric peptide of the present invention can be interchanged arbitrarily. Therefore, although each epitope peptide in the chimeric peptide is represented by HBsAg ^19-33 -HBcAg ^85-140 -epitope peptide TT ^580-599 -β5 epitope peptide-β9 epitope peptide-β8 in the embodiment of the present invention The sequence of epitope peptide-β5 epitope peptide-β9 epitope peptide-β8 epitope peptide is sequentially connected, but those skilled in the art can understand that the positions of any two or more epitope peptides can still be exchanged to obtain a useful chimeric peptides.

In a preferred solution, hydrophilic fragments should be inserted between the epitope peptides to enhance the hydrophilicity of the chimeric peptides and at the same time achieve a suitable length suitable for expression in a biological expression system. In a more preferred embodiment, two or more epitope peptides preferably have a hydrophilic amino acid composition consisting of 6-15 (more preferably 6-10, most preferably 6-8) Water-based spacer. Flexible flexible linkers, such as β-turn peptides or Gly-Gly-Gly-Gly-Ser (GGGGS) repeat sequences, can also be inserted between the epitope peptides to improve the protein molecular conformation of the chimeric peptides. Such a linker is preferably up to 30 amino acids in length and serves to separate the epitope peptides in the chimeric peptide from each other to avoid incorrect folding and inactivation of the chimeric peptide.

In another preferred embodiment, the peptide has the amino acid sequence shown in SEQ ID NO:1.

The second aspect of the present invention provides an isolated nucleic acid sequence, characterized in that it encodes the above-mentioned isolated polypeptide. In a preferred embodiment, the nucleic acid sequence has the nucleic acid sequence shown in SEQ ID NO:2. In the present invention, the term "nucleic acid" refers to deoxyribonucleic acid or ribonucleic acid and polymers thereof in single- or double-stranded form. Unless specifically limited otherwise, the term includes nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. The term "nucleic acid" is used interchangeably with gene, cDNA and mRNA. In addition, the nucleic acid sequences of the invention may be modified to use codons that are preferred for a particular cell class. For example, in order to improve expression in Escherichia coli, some amino acid codons can be converted to codons preferred by Escherichia coli. The nucleic acid sequence of the present invention can be obtained by methods well known to those skilled in the art, for example, it can be obtained by artificial synthesis, chemical splicing, or PCR amplification according to the sequence disclosed in the present invention.

Another aspect of the present invention provides an expression vector, which contains the above sequence and an expression control sequence operably linked to the sequence. The present invention also provides host cells transformed or transduced by the expression vector. The expression vector can be obtained by various methods well known in the art, and the above nucleotide sequences are inserted into a suitable expression vector by selecting appropriate restriction sites, so that they are operatively connected with the expression control sequence in the expression vector. "Expression control sequence" generally refers to a sequence involved in controlling the expression of a nucleic acid sequence. Expression control sequences include a promoter and a termination signal operably linked to a nucleic acid sequence of interest. They also generally include sequences required for proper translation of the nucleic acid sequence. "Operatively linked" means that certain parts of a linear DNA sequence are capable of affecting the activity of other parts of the same linear DNA sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. The expression vector used in the present invention can be various commercially available expression vectors known to those skilled in the art, such as pET11C bacterial expression plasmid.

Then, an appropriate host cell is transformed with the expression vector obtained above. "Host cells" can include prokaryotic cells and eukaryotic cells. In the present invention, preferred host cells are prokaryotic host cells, such as Escherichia coli, Bacillus subtilis and the like. The term "transformation" as used herein refers to direct introduction of an expression vector containing a nucleic acid of interest into a host cell by methods well known to those skilled in the art. Transformation methods vary depending on the host cell type and generally include: electroporation; transfection with calcium chloride, DEAE-dextran, or others; microprojectile bombardment; lipofection; infection and other methods (see Sambrook et al. Molecular Cloning Laboratory Guide, 2nd Edition, 1989). Therefore, another aspect of the present invention also provides host cells transformed with the above expression vectors.

Another aspect of the present invention provides a method for preparing the above-mentioned isolated polypeptide, characterized in that the method comprises: a) obtaining the nucleic acid sequence shown in SEQ ID NO: 2 by chemical synthesis and splicing; b) combining step a ) inserting the obtained nucleic acid sequence into an expression vector so that it is operatively connected with the expression control sequence; c) transforming the host cell with the expression vector described in step b); d) cultivating the obtained peptide obtained in step c) under conditions suitable for the expression of the peptide host cells; and e) separating and purifying the peptides.

Another aspect of the present invention provides a vaccine composition, which contains the above isolated polypeptide and a pharmaceutically acceptable carrier. The vaccine composition can be used for treating tumors and/or human contraception. As used herein, the term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans. The "pharmaceutically acceptable carrier" used herein should be compatible with the polypeptide of the present invention, that is, it can be blended with it without greatly reducing the effect of the composition under normal circumstances. Suitable carriers are usually large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive of virus particles. These vectors are well known and readily selected by those of ordinary skill in the art. The vaccine composition of the present invention can be made into various dosage forms according to needs, and can be administered by a doctor according to factors such as patient type, age, body weight, general disease condition, and administration method to determine a beneficial dosage for the patient. Vaccines can be administered in combination with other immune modulators or immune adjuvants.

Another aspect of the present invention provides an isolated polypeptide sequence consisting of HBsAg ^19-33 , HBcAg ^85-140 and TT ^580-599 (composed of the amino-terminal 92 amino acid residues of SEQ ID NO: 1). This vector can be used to design and construct recombinant multi-epitope chimeric peptides or their (their) coding genes of other target antigens. In the coding nucleotide sequence of the former T-cell epitope peptide, a Taq I (TCGA) restriction endonuclease site is reserved in the middle of the TT5 ^80-599 epitope, so other target antigens have multiple B-cell Epitope tandem gene coding fragments only need to simultaneously synthesize the 11 amino acid base fragments of the second half of the TT epitope at its 5'-end, and the target whole gene fragment splicing can be completed at this Taq I site.

The present invention provides a coding hCGβ doubling six linear B-cell epitopes (-β5 epitope peptide-β9 epitope peptide-β8 epitope peptide-β5 epitope peptide-β9 epitope peptide-β8 epitope peptide) and outer Synthetic gene (CP22) of 6 Th-cell epitopes (including two broad-spectrum epitopes of HBsAg ^19-33 and TT ^580-599 ) and its recombinant expression plasmid. The constructed pET11c-CP22 recombinant plasmid can express the chimeric peptide CP22 protein in the form of inclusion body in Escherichia coli. Compared with the previous design of two types of hCG CPs antigens, CP1 and CP10, CP7 and CP11, which only contain a single set of three β-epitope half-molecule peptides (patent application numbers: 03115893.5 and 03115894.3, both of which are incorporated herein by reference), The present invention realizes the collinear connection of doubling 6 target antigen epitopes and T-cell epitope peptides through genetic engineering, and the expressed CP22 chimeric peptides increase the purpose by deleting the hydrophobic amino acid residues at both ends of each epitope peptide. The CP22 protein is hydrophilic and has a larger molecular weight (favorable for preparative polyacrylamide gel electrophoresis purification), especially the results of animal immunization experiments reveal that its antigenicity and immunogenicity are compared with those that only incorporate 3 target epitopes. The CP antigen is better, that is, only using aluminum salt adjuvant to immunize rabbits and 3 different inbred mouse strains can generate high-level antibody responses, and 3 kinds of antibodies against each epitope can be detected in the antiserum produced , compared with the control standard hCGβ-CTP synthetic peptide/DT, the ability to neutralize hCG in vivo was also significantly improved. Therefore, it lays the foundation for the development of hCG biosynthetic peptide immunogens with better immunogenicity and a wide range of immune responses in immunized populations with different genetic backgrounds. The uniquely designed hCG CP22 antigen/encoding gene can be expressed in Escherichia coli, so it is expected to be possible to obtain hCG recombinant multi-epitope CP vaccine recombination and recombination for the development of new optimized and inexpensive and constant.

Description of drawings

Figure 1 is the SDS-PAGE analysis of hCG recombinant multi-epitope chimeric peptide CP22 expression and purified protein, in which the black arrow indicates the CP22 expressed protein band.

Figure 2 is the Western blot identification of hCG recombinant multi-epitope chimeric peptide CP22 expression and purified protein, where M is the protein molecular weight standard, 1 is 8Mol urea dissolution induced CP22 engineering bacteria inclusion body protein sample, 2 is 8Mol urea dissolution induced CP22 Engineering bacteria inclusion body protein samples, 3 is the purified CP22 protein sample, 4 is the induced CP22 engineered bacteria protein sample, and 5 is the uninduced CP22 engineered bacteria protein sample.

Figure 3 is the detection of the antibody response level after CP22 active immunization of rabbits. The average antibody titer was determined by collecting antiserum on the 7th day after active immunization of rabbits (N=6) with purified CP22 protein (0.5 mg each time) adsorbed by aluminum salt adjuvant (Alum) four times. In the ELESA method, the hCGβ subunit was used as the antigen, and the antibody titer was expressed by the highest dilution. Antigen controls were standard hCGβ-CTP/DT and CP12 protein combined with only 3 B-cell epitopes, and adjuvant control was Freund's adjuvant (CFA).

Figure 4 shows the detection of antibody response levels after active immunization of mice with CP22. The average antibody titer was measured by collecting antiserum on the 7th day after active immunization of three different inbred strain mice (N=4) with purified CP22 protein (0.1 mg each time) in Webster's adjuvant for four times. In the ELESA method, the hCGβ subunit was used as the antigen, and the antibody titer was expressed by the highest dilution.

Figure 5 is the identification of the generation of each epitope antibody in the antiserum collected after CP22 active immunization of rabbits, wherein 1 is the total protein sample of engineered bacteria that does not express streptavidin (Stv)-single epitope (β5BCE) fusion protein, and 2 is Containing the total protein sample of engineered bacteria expressing Stv-β5BCE fusion protein, 3 is the total protein sample of engineering bacteria expressing Stv-β9BCE fusion protein, 4 is the total protein sample of engineered bacteria expressing Stv-β8BCE fusion protein, 5 is the protein molecular weight standard.

Figure 6 is the identification of the generation of each epitope antibody in the antiserum collected after CP22 actively immunized Balb/C mice, wherein 1 is the protein molecular weight standard, 2 is the total protein sample containing the engineering bacteria expressing the Stv-β8BCE fusion protein, and 3 is the sample containing The total protein samples of engineering bacteria expressing Stv-β9BCE fusion protein, 4 are total protein samples of engineering bacteria expressing Stv-β5BCE fusion protein, and 5 are total protein samples of engineering bacteria not expressing Stv-β5BCE fusion protein.

Figure 7 is the identification of the production of each epitope antibody in the antiserum collected after CP22 actively immunized C3H mice, wherein 1 is the protein molecular weight standard, 2 is the total protein sample containing the engineering bacteria expressing the Stv-β8BCE fusion protein, and 3 is the sample containing the expression Stv - β9BCE fusion protein engineering bacteria total protein sample, 4 is the engineering bacteria total protein sample containing the expression Stv-β5BCE fusion protein, 5 is the engineering bacteria total protein sample not expressing the Stv-β5BCE fusion protein.

Figure 8 is the identification of the production of each epitope antibody in the antiserum collected after CP22 actively immunized C57 mice, wherein 1 is the protein molecular weight standard, 2 is the total protein sample of engineering bacteria that does not express the Stv-β5BCE fusion protein, and 3 is the total protein sample containing the Stv-β5BCE fusion protein. - β5BCE fusion protein engineering bacteria total protein sample, 4 is the engineering bacteria total protein sample containing the expression Stv-β9BCE fusion protein, 4 is the engineering bacteria total protein sample containing the expression Stv-β8BCE fusion protein sample.

Figure 9 is the determination of the neutralization performance of rabbit anti-CP22 antiserum in the uterus weighing test. After injecting hCG plus different dilutions of rabbit anti-hCG CP22 antiserum once a day for 3 consecutive days, weigh the uterus weight of immature mice, and compare with hCG plus rabbit anti-standard hCGβ-CTP/DT vaccine antiserum and hCG plus rabbit anti-hCG CP12 Incorporating 3 B-cell epitopes) compared with the antiserum control group, the ability of rabbit anti-hCG CP22 antiserum to neutralize hCG was significantly improved ( ^▲ p<0.001, ^▲▲ p<0.005).

Figure 10A-E shows the cell epitope sequence and its amino acid sequence of each hCG chimeric peptide, wherein Figure 10A is the T- and B-cell epitope sequence and its amino acid sequence in the hCG chimeric peptide CP1; Figure 10B The sequence of T- and B-cell epitopes in hCG chimeric peptide CP10 and its amino acid sequence are shown; Figure 10C shows the sequence of T- and B-cell epitopes in hCG chimeric peptide CP7 and its amino acid sequence; Figure 10D shows the sequence and amino acid sequence of T- and B-cell epitopes in the hCG chimeric peptide CP11 (the hCGβ38-57 peptide segment contains the β5 epitope in the above four hCGCP chimeric peptide sequences, and the hCGβ111-145 peptide segment containing β9 and β8 epitopes); FIG. 10E shows the sequence of T- and B-cell epitopes and their amino acid sequences in the hCG chimeric peptide CP22 of the present invention.

Detailed ways

The purpose of the present invention is to provide a coding hCGβ 3 linear B-cell epitopes (doubled) and exogenous 6 Th-cell epitopes (including two broad-spectrum epitopes of HBsAg ^19-33 and TT ^580-599 ) Splicing synthetic genes (CP22) and their recombinant expression plasmids. All the constructed pET11c-CP22 recombinant plasmids can express the chimeric peptide CP22 protein in the form of inclusion bodies in Escherichia coli. The present invention realizes the linear connection of hCGβ cyclic peptide and hCGβ-CTP with better immunogenicity through genetic engineering, and the expressed hCG chimeric peptide CP22 has enough exogenous broad-spectrum or haplotypes incorporated into its N-terminus. Therefore, it lays the foundation for the development of hCG biosynthetic peptide vaccines that have strong immunogenicity and can generate a wide range of immune responses in immunized populations with different genetic backgrounds. The uniquely designed hCG chimeric peptide CP22 (see SEQ.NO1 for the amino acid sequence) and its coding gene (see SEQ.NO2 for the DNA base sequence) can be expressed in Escherichia coli, and the results of animal immunity experiments prove that: CP22 multi-epitope chimerism Peptide antigen is not coupled with exogenous macromolecular protein carrier and can generate strong immune response in rabbits and mice only with aluminum salt adjuvant; the antigen can produce the same in 3 different inbred strains of mice anti-CP22 antibodies in rabbits and mice were detected against the three linear epitopes of hCGβ in the designed molecule; compared with the standard hCGβ-CTP/DT chemically synthesized peptide vaccine, anti-hCG CP22 antibodies The performance of serum neutralizing natural hCG was significantly enhanced (statistically significant). Therefore, hCG CP22 recombinant multi-epitope chimeric peptide may become a new antigen for the development of human contraception and tumor therapy.

According to the principles of immunology and the status quo of hCG vaccine research, especially the main obstacles to its popularization and application, for example, how to obtain hCG vaccine antigen cheaply and constantly? How to increase the neutralization performance of hCGβ-CTP synthetic peptide antiserum on the basis of specificity? Can the use of macromolecular protein carriers and special adjuvants that provide Th cell epitopes required for immune responses in vivo be avoided to facilitate the production of vaccine preparations and reduce production costs? In addition, there is also the problem of "vaccine names", that is, how to avoid the MHC genetic restriction, and the effective immune response rate in the immunized population of women who want to avoid pregnancy, including patients with hCG hormone-dependent malignant tumors? We designed a new optimized hCG chimeric peptide immunogen CP22 to solve the above problems, which consists of 3 linear ratio cellular epitopes (doubled) and 6 exogenous epitopes including 2 broad-spectrum epitopes in hCGβ Th cell epitope composition.

The present invention provides a uniquely designed CP22 multi-epitope chimeric peptide splicing combination sequence, that is, HBsAg ^19-33 -HBcAg ^85-140 -TT ^580-599 -β-turn peptide-hCGβ ^42-54 -hydrophilic fragment in hCGβ- hCGβ ^109-122 -hCGβ ^132-145 -hydrophilic fragment in hCGβ -hCGβ ^42-54 -hCGβ ^111-121 -hCGβ ^133-140 . The epitope fragments of CP22 in SEQ.NO1, except for the β-turn peptide (Leu-Ser-Pro-Gly), are selected from the vaccine components that have been marketed in clinical applications or have passed human clinical trials without safety problems. The hCGβ epitope peptide and hydrophilic fragment encoding gene sequence was based on the cloned hCGβ cDNA sequence (Fiddes JC and Goodman HM. The cDNA for the β-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3′untranslated region. Nature 286:684-687, 1980), in which part of the amino acid codons were changed to codons preferred by Escherichia coli.

The β5 ^45-52 antibody neutralizing epitope and the β9 ^113-116 and β8 ^137-144 specific epitopes in the HCG CP22 chimeric peptide were selected based on the following published literature: Stevens VC, et al. The identification of peptidesequences of human chorionic gonadotropin containing a conformational epitope. Immunol Litters 12:11-18, 1986; Dirnhofer S, et al. The molecular basis for epitopes on the freeβ-subunit of human chorionic gonadotrophin(hCG), its carboxyl-terminal peptide- and the hC core fragment. J Endocrinol 141:153-162, 1994. Considering that biosynthetic peptides need a certain length, and that the expression products are convenient for SDS-PAGE electrophoresis detection, and that appropriately increasing the length of synthetic peptides is also conducive to enhancing immunogenicity, in the design of the target CP molecule, a band with both ends was used. Relatively hydrophilic amino acid residues. In addition, in order to enhance the hydrophilicity of the target molecule and/or improve the conformation of the protein molecule, the hydrophilic fragment (8 or 6 residues, individual residues have been adjusted) and β Corner peptide.

The hCG CP22 chimeric peptide of the present invention is spliced through the gene fragments encoding the above-mentioned epitopes or peptide fragments, and an Ecor I-Nde I enzyme cleavage site and an ATG initiation codon are added at its 5' end, and at its 3' After the TAA stop codon and BamH I restriction site are connected at the end, the bacterial expression plasmid pET11c, which can be induced by IPTG, is recombined with the complete target chimeric peptide reading frame, and finally in the low-cost and easy-to-operate Escherichia coli Expression of CP22 was achieved. The expression product was identified by Western blotting test with the monoclonal antibody OT3A which can recognize hCGβ ^133-139 sequence, which confirmed the specific expression of the designed and constructed hCG CP22 chimeric peptide accounting for about 1% of the total cell protein.

In the recombinant multi-epitope hCG CP22 constructed in the present invention, the selection of the six cell epitopes at the N-terminal is based on the following public information:

1. It is widely known that HBsAg is a powerful immunogen that can induce autoantibody production, indicating that it itself has a strong Th-cell epitope. HBsAg ^19-33 was identified as a "broad-spectrum" T-cell epitope recognized by different HLA-DR and -DQ haplotypes. Greenstein JL et al. reported that this peptide is a chimeric peptide synthesized collinearly with the HIV-1 envelope gp120 third variable region domain (V3 loop, which can guide the effective neutralizing antibody region), and it can be absorbed only by aluminum salt adjuvant. Anti-V3 cyclic peptide antibodies were induced in all 6 different inbred mouse strains (A universal T cellepitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120. J Immunol 148: 3970-3977, 1992) ;

2. HBcAg is also a powerful immunogen in the marketed vaccines. Each T cell epitope present in its 85-140 peptide has also been identified (Tiollais P, et al. Biology of hepatitis B virus. Science, 1981; 213: 406-411; Milich DR, et al. Hepatitis B synthetic immunogen comprised of nucleocapsid T-cell sites and an envelope B-cell epitope. Proc Natl Acad Sci USA. 1988; 85: 1610-1614). It is now clear that HBcAg ^85-100 is H-2 ^d , HBcAg ^100-120 is H-2 ^f and H-2 ^q , HBcAg ^120-131 is B10.S(H-2 ^s ), HBcAg ^129-140 is B10( H-2 ^b ), and HBcAg ^120-140 is the MHC class II restricted T helper cell epitope of H-2 ^s,b . It is clear that at least four Th-cell epitopes are contained in the HBcAg ^85-140 peptide. The selection of the HbcAg peptide consisting of 57 amino acid residues is not only to improve the broad-spectrum T cell stimulation of the constructed chimeric peptide, so as to enhance the molecular adjuvant effect of the T cell epitope peptide, but also to achieve The biosynthesis of peptides requires a certain length of consideration;

3. As we all know, the definition of broad spectrum is relative. Therefore, in order to generate an immune response rate as close to 100% as possible in the immune population with divergent genetic backgrounds, another broad-spectrum Th- Cell epitope---TT ^580-599 (Ho PC, et al. Identification of two promiscuous T cell epitopes from tetanus toxin. Eur J Immunol, 1990; 20: 477-483. Kaumaya PTP, et al. Peptide vaccines incorporating a 'promiscuous' T-cell epitope bypass certain haplotype restricted immune responses and provide broadspectrum immunogenicity. J Mol Recog, 1993; 6: 81-94).

The present invention utilizes recombinant DNA technology to express hCG multi-epitope chimeric peptide and conducts animal immunological detection.

Specific steps are as follows:

(1) The design of the full-length coding cDNA of hCG CP22 chimeric peptide, connecting the coding gene fragments in sequence, adding the start codon ATG and the previous EcoR I-Nde I sticky enzyme cutting site to the 5'-end Point bases, 3'-terminal double stop codon TAATGA and subsequent BamH I sticky enzyme cutting site bases to form a complete CP22 coding gene (SEQ.NO2);

(2) Decompose the positive and negative strands of the CP22 coding gene into 12 fragments for chemical synthesis, and then splice them by DNA recombination technology;

(3) Cloning and sequencing identification of the full-length coding cDNA of hCG CP22 chimeric peptide;

(4) The construction of pET11c-hCG recombinant expression vector and its expression in Escherichia coli, the expression vector selects pET11c plasmid, uses Nde I and BamH I two kinds of restriction endonucleases from the pBS/CP22 plasmid identified through DNA sequencing The CP22 gene was excised by enzymes, and then recombined into the pET11c plasmid. The recombinant expression plasmid was identified again by DNA sequencing, and then transformed into BL21(DE3)plysS host bacteria respectively;

(5) Separation and purification of the target expression product CP22;

(6) Rabbits and mice of different inbred strains were actively immunized, and the antigenicity and immunogenicity of CP22, as well as its ability to overcome MHC genetic restrictions and its antisera to neutralize hCG were tested.

The recombinant hCG multi-epitope chimeric peptide CP22 provided by the present invention combines all three linear B-cell epitopes in the target antigen hCGβ subunit (a total of 6 double copies, wherein the second β8 epitope is in the order of OT3A monoclonal antibody recognition ^133-139 substitutions) and exogenous proteins including 6 Th-cell epitopes including two broad-spectrum epitopes. Since the recombinantly expressed hCGCP22 immunogen not only retains the two specific B-cell epitopes of β9 and β8 of hCGβ-CTP chemically synthesized peptide, but also incorporates the antibody neutralizing epitope of β5, so the animal antiserum While anti-β9 and β8 antibodies reflecting its specificity could be detected, its ability to neutralize hCG was significantly improved by the presence of β5 antibody. These relevant experimental evidences indicate that the present invention can form a new hCG tumor immunotherapy and/or human immune contraceptive synthetic peptide vaccine preparation method and biological products. The 6 T helper (Th-) cell epitope tandem peptide moieties in CP22 selected 1 T-cell epitope HBsAg ^19-33 in the hepatitis B surface antigen and 4 T-cell epitopes in the hepatitis B core antigen. The peptide segment HBcAg ^85-140 and a T-cell epitope TT ^580-599 in tetanus toxoid. T-cell epitope peptides combined in tandem can act as molecular adjuvants to mobilize T cell responses in the immune system to secrete Th2 cytokines, so such CP immunogens no longer need to interact with exogenous Th-cell epitopes. Macromolecular protein coupling and special immune adjuvants are used, and at the same time, it is convenient for the production of vaccine preparations (it only needs to be adsorbed by human available aluminum salt adjuvants). The two epitopes of HBsAg ^19-33 and TT ^580-599 are characterized in that they are strong and broad-spectrum T-cell epitopes. The combination of the two in one recombinant multi-epitope chimeric peptide helps to overcome the common problem of "vaccine family names" in vaccine development. (major histocompatibility complex, MHC) genetic backgrounds are different in the immunized population to produce the maximum effective immune response rate close to 100%.

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 and are not intended to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are all according to conventional conditions such as the conditions described in "Molecular Cloning: Laboratory Manual" (Third Edition) (Cold Spring Harbor Laboratory Press) of Sambrook et al. conditions recommended by the manufacturer.

Example

Materials and methods

1. Restriction enzymes EcoR I, Sal I, Nde I, and BamH I are products of Boehringer Mannheim in the United States, T4 DNA ligase is a product of British Biolabs, T4 polynucleotide kinase is a product of New England Biolabs in the United States, and Taq I DNA polymerase is a product of Products of the State Key Laboratory of Genetic Engineering of Fudan University, IPTG is a product of Promega in the United States, protein low molecular weight standards and alkaline phosphatase-linked goat anti-mouse secondary antibody (IgG/HRP) are products of Huamei Bioengineering Company, 5-bromo-4-chloro -3-Indole-β-D-galactoside (X-gal) and isopropylthio-β-D-galactoside (IPTG) are products of Sigma Company in the United States, monoclonal antibodies that recognize hCG β ^133-139 sequence OT3A was kindly provided by Organon Technika, Netherlands.

2. Cloning and sequencing The vector pBluescript KS (PBS) was purchased from Stratagene, and the expression vector pET11c was a product of Novagen. Escherichia coli TG1 and BL21(DE3) are strains preserved by the State Key Laboratory of Genetic Engineering, Fudan University.

3. QIAprep SPIN Plasmid Extraction Kit, QIAquick PCR Purification Kit and QIAquick Gel Recovery Kit are products of QIA, Germany.

4. The 24 (twelve pairs) nucleotide base fragments of the positive and negative strands of the designed CP22 coding gene were synthesized by Shanghai Sangong Company.

5. Experimental animals: New Zealand male white rabbits (body weight 2.0±0.5 kg) were provided by SIPPR-BK Lab Animal Ltd Co. (Shanghai). 6-9 weeks old Balb/C(H-2 ^b ), C3H/hej(H-2 ^k ) and DBA/1(H-2 ^q ) female mice (18-20g) were purchased from SIPPR-BK Lab Animal Ltd Co. (Shanghai) and Shanghai Experimental Animal Center, Chinese Academy of Sciences. hCG, hCG β subunit and Freud's adjuvant were purchased from Sigma, USA, and aluminum salt adjuvant was purchased from Shanghai Institute of Biological Products. The standard WHO hCGβ-CTP/DT synthetic peptide vaccine was donated by Professor Stevens of Ohio State University.

The specific steps of preparation are as follows:

1. Design of the complete coding gene of hCG CP22

The amino acid sequence sequence of foreign protein T- and target antigen double B-cell epitope and other fragments in hCG chimeric peptide CP22 is shown in SEQ ID NO: 1.

The design of the full-length cDNA of hCG CP22 was completed with computer assistance, and the codons preferred by Escherichia coli were basically selected in the reading frame, according to the published coding gene fragments of each T-cell epitope peptide and B-cell epitope peptide Panel design. After searching by PC-GENE software, the constituent bases of individual codons were adjusted to eliminate possible positive and negative repeat sequences, including the occurrence of base complementarity in overlapping regions between fragments and inappropriate restriction sites. At the 5'-end of the hCG CP1 gene reading frame, the start codon ATG and the previous EcoR I-Nde I sticky restriction site were added, and the double stop codon TGATAA and the following were added at its 3'-end Bam H

I sticky enzyme cutting site.

2. Splicing of the complete hCG CP22 gene

The CP22 gene with a full length of 562 base pairs (EcoR I and BamH I cohesive ends are reserved at both ends of the positive and negative strands) was divided into 24 oligonucleotide fragments ranging in length from 31-mer to 57-mer for synthesis. After synthesis, add ddH ₂ O to dilute each fragment to 20pmol/μl, and first conduct 16% polyacrylamide denaturing gel electrophoresis to identify the purity, and then take 16μl each (excluding the two fragments at the 5′-end of the positive and negative strands) in 0.5μl 10U/μl The 5'-terminal phosphorylation of 18 fragments was catalyzed by μld T4 polynucleotide kinase (plus 2 μl of 10mmol/L ATP). Subsequently, the corresponding fragments of the positive and negative strands are annealed and annealed in pairs, and then the adjacent DNA fragments are connected in pairs by enzymatic reactions. The final complete gene ligation reaction solution was separated by 5% non-denaturing polyacrylamide gel electrophoresis, ethidium bromide (EB) staining, and then the full-length target gene fragment was cut under long-wavelength ultraviolet light against molecular weight standards, and crushed with ""Immersionmethod" to recover DNA.

3. Cloning and sequencing of hCG CP22 gene

The CP22 gene fragment at the cohesive ends of EcoR I and BamH I at both ends of the reserved gene is directly inserted into the pBS cloning sequencing vector that has also been digested by EcoR I and BamH I through DNA recombination, and then transformed into Escherichia coli TG1 host bacteria with the enzyme ligation reaction solution. Finally, the white colonies that may be recombinant clones were screened on the LB medium plate coated with 20% X-gal and 100mmol/L IPTG. Several extracted recombinant plasmids were identified by preliminary enzyme digestion, and the gene base sequence was analyzed on the PAGE gel of the ABI373A automatic DNA sequencer. Finally, determine the positive clones whose inserted gene sequence is consistent with the design.

4. Construction of pET11c/hCG CP22 recombinant expression vector and its expression in Escherichia coli

As the expression vector, pET11c plasmid with strong T7 promoter which can be induced by IPTG was selected. First use NdeI and BamH two restriction enzymes to excise the CP22 gene fragment from the pBS/CP1 plasmid identified by DNA sequencing, and then use T4 DNA ligase to recombine it into the NdeI and BamHI sites of the pET11c plasmid. The recombinant plasmids were identified by DNA sequencing again and transformed into protease-deficient host bacteria BL21(DE3)plysS respectively. The induced expression conditions of engineering bacteria BL21(DE3)/pET11c-CP22 are as follows: inoculate the target engineering bacteria in 50ml LB medium (250ml shake flask) containing 50μg/ml ampicillin and 34μg/ml chloramphenicol. Shake culture at 37°C for 3-4 hours to make the OD ₆₀₀ value of the culture reach 0.5, take 3ml of the initial culture and inoculate it into 500ml LB medium (2000ml shake flask) containing 500μg ampicillin and 340μg chloramphenicol, and shake and ferment at 37°C Make the OD ₆₀₀ value of the culture reach 0.8-1.0 in 3-4 hours, then add IPTG (final concentration 1.0 mM) for induction, and continue fermentation for 4-5 hours. The bacterial solution was centrifuged at 5000 rpm for 15 minutes at 4°C to collect the bacterial pellet.

5. SDS-PAGE analysis and Western blot identification of the target expression product.

The induced bacterial precipitate was resuspended in the bacterial lysate at the ratio of adding 5ml of solution per gram of wet bacteria, ultrasonically disrupted the bacteria for 0.5-1 hour, collected the bacterial precipitate by centrifugation at 4°C, and then added 50ml containing 0.5% Triton The lysate was homogenized by ultrasonication, then centrifuged at 4°C to collect the precipitate, washed with 50ml 1mol/L urea, suspended by ultrasonication with 25ml 8mol/L urea, and centrifuged to obtain the supernatant for SDS-PAGE analysis. Repeat the loading of the other half of the gel for electroblotting, and the target expressed protein on the nitrocellulose membrane is reacted with the specific monoclonal antibody OT3A (primary antibody) and horseradish peroxidized alkaline phosphatase-labeled goat anti-mouse secondary antibody Afterwards, the color was developed with DAB solution.

6. Separation and purification of the target expression product CP22

Preparative SDS-PAGE method [Zou Yongshui, Xu Wanxiang et al.: Preparation of recombinant human chorionic gonadotropin chimeric peptide 12 by polyacrylamide gel electrophoresis. Acta Biochemistry and Biophysics, 2002; 34(5): 671-674 ] One-step purification of CP22 expression protein, 0.1-0.5 mg of target protein with electrophoretic band uniformity higher than 95% can be obtained per liter of E. coli culture medium. Protein purity was detected by SDS-PAGE method.

7. Immunological detection of animals actively immunized with hCG CP22 purified protein

(1) Active immunization of New Zealand white rabbits (N=6): Active immunization adopts multi-point injection on the back. 0.5 mg of CP22 protein dissolved in 0.5 ml of normal saline plus 0.5 ml of complete Freudian adjuvant (CFA) is fully emulsified or ground and adsorbed with 1 ml of aluminum salt adjuvant, and each injection is 1 ml each time. Ten days later, 0.5 mg of CP immunogen emulsified with 0.5 ml of incomplete Fresund's adjuvant (IFA) was injected. Every other week, the immunization was strengthened twice in the forefoot muscles of the rabbits. hCG

The β-CTP/DT vaccine was only adjuvanted with CFA. In addition, only CFA or aluminum salt adjuvant was injected in the control group.

(2) Active immunization of three inbred strains of mice with different -2 haplotypes: In the first week, multi-point injection of 0.1 mg of CP22 protein antigen absorbed with 0.1 ml of CFA adjuvant in the hip and neck of the mouse 10 days later, the CP22 immunogen emulsified with 0.1ml IFA and dissolved in 0.5ml normal saline was used for immunization again. Every other week, the immunization was boosted twice in the forefoot muscles of the mice.

(3) ELESA antibody titer determination: ELESA method was used for the determination of antibody titer. After the 4th booster immunization, blood was collected from the rabbit's ear vein every other week to detect the serum antibody titer. From the 3rd week after the first immunization, blood was collected from the tail vein of the mice, and blood was collected every week or every two weeks thereafter to determine the serum antibody titer. In the ELESA method, the hCGβ subunit of Sigma Company of the United States was used as the antigen to coat the microtiter plate, and serially diluted antiserum was added as the primary antibody, goat anti-rabbit/mouse IgG-HRP was used as the secondary antibody, and the H ₂ O ₂ -OPD system displayed color, finally in the ELX 800

Universal Microplate Reader (Bio-TEK Instruments, Inc.) was used for determination.

(4) Detection of each BCE antibody production in immune serum: We have previously constructed streptavidin fusion proteins with single epitopes of β5, β9 and β8 that can be highly expressed in Escherichia coli [Xu Wanxiang et al.: human chorionic gonadotropin Expression and purification of β subunit single B-cell epitope (β5, β9 or β8) fusion protein. Acta Bioengineering, 2004; 20(1): 49-53]. Take the total protein of their induced engineered bacteria as the sample (antigen) for SDS-PAGE, then electrotransfer the expressed protein to a nylon membrane, stain with ponceau briefly, and punch holes at both ends of the fusion protein to be expressed, and then wash with water to elute the color , and finally use the highest titer rabbit/mouse antiserum as the primary antibody for western blot analysis.

(5) Mouse uterus weighing experiment: select 18-25 day old Balb/C female mice, use 200ng of hCG standard (purchased from Sigma, USA), and make it compare with CP12, CP22 and control β-hCG respectively Rabbit antiserum stock solution of CTP:DT and diluted antiserum of 1:10 and 1:50 were intraperitoneally injected for 3 consecutive days, and sacrificed 24 hours after the last injection, and the weight of the uterus of mice was weighed. Negative and positive control groups were injected with normal saline and hCG, respectively.

The research results show that the present invention has successfully spliced, synthesized and constructed the hCG chimeric peptide CP22 artificial gene and the pET11c/CP22 recombinant expression plasmid, which can express CP22 in the form of inclusion bodies after transforming the host Escherichia coli BL21(DE3)plysS protein, and was verified by Western blot experiments (Figure 1 and Figure 2).

The results of SDS-PAGE analysis show that the expression level of hCG chimeric peptide CP22 in E. coli is about 1%, and 0.5 mg of the target with electrophoretic uniformity higher than 90% can be harvested per liter of engineering bacterial culture solution by preparative SDS-PAGE method protein (Figure 1 and Figure 2).

Animal immunology experiments also obtained the expected results: 1. Since the designed CP22 chimeric peptide incorporates 6 exogenous T-cell epitopes, only human-available aluminum salt adjuvants are used to actively immunize rabbits and mice, and CP22 can produce The level of antibody response was very close to that of Freund's complete adjuvant (Fig. 3 and Fig. 4); 2. The presence of the incorporated β5, β9 and β8 antibodies could be detected in both rabbit and mouse anti-CP22 antisera (Figure 5-Figure 8), it shows that each epitope in the CP22 synthetic peptide is presented undamaged during the antigen processing of the immune system in vivo; 3, because two broad-spectrum T-cell epitope peptides are selected -Cell epitopes, in the three different inbred strains of immunized mice all produced very similar levels of antibody responses, including the presence of antibodies to each epitope (Figure 4, Figure 6-Figure 8), suggesting that the CP22 antigen was initially It has the ability to overcome the genetic limitation of MHC; 4, due to the incorporation of the β5 antibody neutralizing epitope in the CP22 recombinant multi-epitope chimeric peptide, it is different from the standard β-hCG containing only two specific B-cell epitopes of β9 and β8 Compared with CTP:DT chemically synthesized peptide vaccine, its ability to neutralize hCG in vivo was statistically significantly improved (Table 1 and Figure 9).

Table 1 Determination of neutralizing hCG performance of rabbit anti-CP22 antiserum in uterine weighing test

Group Uterine weight (mg/g body weight) Dilution 1:1 1:10 1:50 Saline control hCG control hCG+CP12 antiserum hCG+CP22 antiserum hCG+CTP antiserum 9.90±0.6228.88±1.4112.30±0.37^▲10.04±0.29^▲15.16±0.90^▲▲ 9.90±0.6228.88±1.4114.00±0.24^▲11.48±0.27^▲16.68±0.53^▲▲ 9.90±0.6228.88±1.4115.46±0.43^▲▲14.46±0.91^▲19.12±0.87^▲▲

Obviously, the construction of the hCG chimeric peptide CP22 chimeric gene and the acquisition of its expressed protein, especially the good animal immunological test results, provide a new and optimized method for the development of hCG hormone-dependent malignant tumor therapy and human fertility regulation targets. The hCG synthetic peptide vaccine has laid the foundation and is also of great significance in the field of developing other synthetic peptide vaccines against viruses and/or parasites.

Although specific examples have been described herein, it will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes which are within the scope of this invention.

sequence listing

<110>Shanghai Family Planning Research Institute

Fudan University

<120> Molecular design and preparation method of human chorionic gonadotropin recombinant multi-epitope chimeric peptide CP22 antigen

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Claims (7)

1. an isolated polypeptide is characterized in that, it is human chorionic gonadotrophin recombinant multi-epitope chimeric peptide CP22, and its aminoacid sequence is shown in SEQ ID NO:1.

2. an isolated nucleic acid sequences is characterized in that, the described polypeptide of its coding claim 1.

3. nucleotide sequence according to claim 2 is characterized in that, this nucleotide sequence is shown in SEQ ID NO:2.

4. an expression vector is characterized in that, the expression regulation sequence that it contains the described nucleotide sequence of claim 2 and links to each other with this series of operations.

5. a host cell is characterized in that, it is transformed by the described expression vector of claim 4.

6. a method for preparing the described isolated polypeptide of claim 1 is characterized in that, this method comprises:

A) method with the chemosynthesis splicing obtains the nucleotide sequence shown in the SEQ ID NO:2;

B) will in the step a) gained nucleotide sequence insertion expression vector it be linked to each other with the expression regulation sequence operability;

C) with the described expression vector transformed host cell of step b);

D) host cell of gained culturing step c under the condition that is fit to described peptide expression); With

E) separation and purification obtains described peptide.

7. vaccine composition, it contains described isolated polypeptide of claim 1 and pharmaceutically acceptable carrier.

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