CN100368436C - Molecular design and prepn process of recombinant polyepitope chimeric peptide CP22 antigen of human chorionic gonadotropin - 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

Antigenic molecular designing of human chorionic gonadotrophin recombinant multi-epitope chimeric peptide CP22 and preparation method thereof

Technical field

The present invention relates to gene engineering technology field, more particularly, the present invention relates to human chorionic gonadotrophin recombinant multi-epitope chimeric peptide CP22 antigen and preparation method thereof.

Background technology

(human chorionic gonadotropin, hCG) another name progesterone are to keep human early stage conceived a kind of glycoprotein hormones to human chorionic gonadotrophin, and it is made of α and two subunits of β.In addition, it also early is accredited as is a kind of carcinomebryonic antigen, it all has expression in many malignant tumours, for example in cancers such as pancreas, lung, knot rectum, uterine cervix, ovary, liver, stomach, can both detect hCG and subunit or segmental existence the (Triozzi P and StevensVC.Human chorionic gonadotropin as a target for cancer vaccines.Oncol Rep 6:7-17,1999).Therefore, hCG is considered to develop the important target antigen of contraception and/or tumor therapeutic vaccine always.As vaccine, the maximum of usefulness are its hCG β subunit that purifying splits from natural hCG and the hCG β carboxyl terminal that contains β 9 and 8 two specific B-cell epitopes of β (carboxyl-terminal peptide, CTP) synthetic 37 peptide antigens so far.Though passed through the clinical II of people phase efficacy test (Talwar GP with sheep LH alpha subunit link coupled hCG β heterodimer (HSD-hCG) vaccine, et al.A vaccine that prevents pregnancy in women.Proc Natl Acad SciUSA, 1994; 91:8523-8536), verify the feasibility of immunological contraception and had milestone significance, but because its some defectives that are difficult to overcome and practical problems are not suitable for applying (Stevens VC.Progress in thedevelopment of human chorionic gonadotropin antifertility vaccines.Am J ReprodImmunol, 1996 by identification; 35:148-155.Talwar GP, Singh Om, Gupta SE, et al.The HSD-hCGvaccine prevents pregnancy in women:feasibility study of a reversible safe contraceptivevaccine.Am J Reprod Immunol, 1997; 37:153-160).For example: purifying fractionation hCG beta antigen is difficult and cost is high; Vaccinogen need with sheep LH alpha subunit and Toxoid,tetanus (TT) or diphtheria toxoid (DT) coupling, and need special adjuvant because of being that the autoantigen immunogenicity is weak, except that increasing greatly the vaccine cost thus, and be difficult to by GMP standard mass production vaccine; Have 20% to be tried that the women does not reply vaccine or invalid response etc. in the efficacy test of clinical II phase of people.Also there is similar problem in hCG β-CTP synthetic peptide vaccine, and the corresponding clinical II phase efficacy test of people is failed smooth implementation always.But, it has still passed through the clinical II of people phase efficacy test (Moulton HM as hCG dependency treating malignant tumor vaccine recently, et al.Active specific immunotherapy with a β-human chorionic gonadotropin peptide vaccine in patients with metastatic colorectalcancer:antibody response is associated with improved survival.Clin Cancer Res, 2002; 8 (7): 2044-2051), the effect that active immunity suppresses tumor growth or prolongs patient lifetime is obvious, thereby has represented the hCG β-application prospect of CTP synthetic peptide vaccine on immunotherapy of tumors.But the superfluous words of need not, real actual persons clinical application also need to overcome solve with DT coupling, the special adjuvant of needs (reduce the vaccine cost and be convenient to preparation production), vaccine specific by force but the antibody that produces among the hCG and poor performance and how to improve and improve problems such as postvaccinal immunne response rate of different genetic background tumour patients and therapeutic efficiency.

On the other hand, the development of chemosynthesis peptide vaccine is placed high hopes always, wish that it can make contributions for human prevention and treatment of diseases, but the still untapped synthetic peptide vaccine that goes out an energy practical application over nearly 40 years is many such as obtaining obviously advantage such as constant and inexpensive relatively private antigen although it has.Cause the reason of this situation to be that mainly (B-cell epitope, BCE) peptide is not enough to produce immune effect completely to the single B-cell epitope of target antigen.However, multi-epitope biosynthesizing (group of also weighing) 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 that those researchs still form for development at present; 2 (4): 95-100) lay a good foundation, some of them research enlightenment collinearity in synthetic peptide is mixed strong and/or " broad spectrum " T-cell epitope (T-cell epitope, TCE with connecting; Sinigaglia F, et al.A malaria T cell epitope recognized in association with mostmouse and human class II molecules.Nature 336:778-780,1998; Panina-Bordignon P, etal.Universally immunogenic T cell epitopes:promiscuous binds to human class II andpromiscuous recognition by T cells.Eur J Immunol, 1989; 19:2237-2242), can play the molecule adjuvant effect, and make purpose chimeric peptide vaccine have performance (the Greenstein JL that crosses over the hereditary restriction of MHC immunne response, 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 pellucida 3peptide vaccine induces antibodies and reversible infertilitywithout ovarian pathology.J Immunol, 1995; 155:2715-2720), thereby need not special adjuvant for capturing simultaneously to solve, improve the immunne response rate in different genetic background crowds, promptly a difficult problem such as vaccine One Hundred Family Names provides the new approaches new way.Be subjected to the restriction of chemical synthesising peptide length, development reorganization (also claiming genetically engineered or biosynthesizing) multi-epitope chimeric peptide (chimeric peptide, CP) be a kind of inevitable choice, but because relevant knowledge and technology limitation, animal immunology research could be expressed and finish to designed peptide chimeric molecules by biology department system report seldom, but only several examples have still represented the feasibility and the development prospect of recombinant multi-epitope synthetic peptide vaccine, improved immunologic function (Sivapurapu N as CP at 3 B-cell epitopes of three glycoprotein of the combined hat monkey ZP of expression in escherichia coli, et al.Efficacy of antibodies against Escherichia coli expressed chimeric recombinant proteinencompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro humansperm-egg binding.Mol Reprod Dev, 2003; 65:309-317), particularly not only need not special adjuvant and just can produce stronger special antibody response in the plasmodium CP immunogen of 12 B-cell epitopes of combination of expressed in insect cells and 6 T-cell helper epitope and 1 broad spectrum T-cell epitope, has ability (the Shi YP that crosses over the MHC restriction but also demonstrate, Hasnain SE, Sacci JB, et al.Immunogenicity and 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 multi-stage Plasmodium falciparummalaria vaccine candidate.Vaccine, 2000,18:2902-2914).

Except above hCG vaccine, the especially enlightenment of the development present situation of hCG β-CTP synthetic peptide vaccine and the new development of chemical synthesising peptide field, the necessity of development reorganization hCG multi-epitope CP is also based on following report, and is promptly compound and with hCG β cyclic peptide ^38-57(include β 5 linear B-cell epitopes ^45-52) and hCG β-CTP ^109-145(include the special linear B-cell epitope of β 9 and β 8 ^{113-116,137-144}) prompting of two kinds of synthetic peptide vaccine experiments, because synthesizing the antibody response level of peptide (all with the DT coupling) 1: 1 mixture of vaccine, hCG β cyclic peptide and hCG β-CTP be significantly higher than both effects of immunity separately, and compare with the antiserum(antisera) of single synthetic peptide vaccine, the antiserum(antisera) neutralization that mixture immunity produces and in conjunction with the ability of target hCG also improve significantly (Stevens VC.Am J Reprod Immunol, 1996; 35:148-155).

Summary of the invention

One aspect of the present invention provides a kind of isolated polypeptide, and this peptide is human chorionic gonadotrophin recombinant multi-epitope chimeric peptide CP22, and it is basically by epitope peptide HBsAg ^19-33, epitope peptide HBcAg ^85-140With epitope peptide TT ^580-599, and two groups of human chorion gonadotrophic hormone beta 5 epitope peptides, β 8 epitope peptides and β 9 epitope peptides are formed.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.Term " isolated polypeptide " is meant that polypeptide of the present invention is substantially free of other albumen, lipid, carbohydrate or other material.Those skilled in the art can come this polypeptide of purifying with the purified technology of protein of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.Term " basically by ... form " refer to also can comprise in this polypeptide other any amino acid, as long as these occurrences of amino acid do not have substantial disadvantageous effect for the biological effect of chimeric peptide of the present invention.As follows, the flexible joint or the corner peptide that strengthen the hydrophilic amino acid fragment of chimeric peptide or improve the protein molecular structure also can be arranged between each epitope peptide in the chimeric peptide of the present invention.

The succession of each epitope peptide that contains in the chimeric peptide of the present invention can be exchanged arbitrarily.Therefore, although in embodiments of the present invention in this chimeric peptide each epitope peptide be with HBsAg ^19-33-HBcAg ^85-140-epitope peptide TT ^580-599The order of-β 5 epitope peptides-β 9 epitope peptides-β 8 epitope peptides-β 5 epitope peptides-β 9 epitope peptides-β 8 epitope peptides connects successively, but those skilled in the art can understand, the location swap of any two or more epitope peptides wherein still can be obtained useful chimeric peptide.

In preferable scheme, should insert the wetting ability fragment between each epitope peptide, to strengthen the wetting ability of described chimeric peptide, be fit to the appropriate length that a biological expression system is expressed to reach simultaneously.In a better embodiment, should have the wetting ability spacerarm that constitutes by 6-15 (be more preferred from 6-10, the best is 6-8) hydrophilic amino acid between two or more epitope peptides.Can also insert the flexible joint of flexing between the epitope peptide, as βZhuan Jiao peptide or Gly-Gly-Gly-Gly-Ser (GGGGS) tumor-necrosis factor glycoproteins etc., to improve the protein molecular conformation of chimeric peptide.This length of said joint should reach 30 amino acid, and its effect is that each epitope peptide in the chimeric peptide is separated from each other, and to avoid that incorrect folding taken place chimeric peptide is lost activity.

In another preferable embodiment, described peptide has the aminoacid sequence shown in the SEQ ID NO:1.

Second aspect of the present invention provides a kind of isolated nucleic acid sequences, it is characterized in that its above-mentioned isolated polypeptide of encoding.In a preferable embodiment, described nucleotide sequence has the nucleotide sequence shown in the SEQ ID NO:2.In the present invention, term " nucleic acid " refers to the polymkeric substance of thymus nucleic acid or Yeast Nucleic Acid and strand or double chain form.Unless special qualification is arranged in addition, this term comprises the nucleic acid of the known analogue that contains natural nucleotide, and this nucleic acid has the bonding properties similar to reference nucleic acid, and carries out metabolism in the mode similar to naturally occurring Nucleotide.Term " nucleic acid " can exchange with gene, cDNA and mRNA and use.In addition, nucleotide sequence of the present invention can be through modifying to use at the preferable codon of special cell classification.For example, in order to improve the expression in intestinal bacteria, the partial amino-acid codon can be transformed to the codon of intestinal bacteria preference.Nucleotide sequence of the present invention can obtain with method well known to those skilled in the art, and for example, can increase according to sequence synthetic disclosed by the invention, chemistry splicing or with the PCR method obtains.

The present invention provides a kind of expression vector on the other hand, the expression regulation sequence that this expression vector contains above-mentioned sequence and links to each other with this series of operations.The present invention also provides the host cell that transforms or transduce through this expression vector.This expression vector can obtain with the whole bag of tricks well known in the art, by select proper restriction site will above-mentioned these nucleotide sequences insertion suitable expression vector in, they are linked to each other with expression regulation sequence operability in the expression vector." expression regulation sequence " is often referred to and participates in the sequence that the control nucleotide sequence is expressed.Expression regulation sequence comprises promotor and the termination signal that links to each other with the target nucleic acid series of operations.They also comprise the suitably required sequence of translation of nucleotide sequence usually." operability links to each other " is meant that some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if promotor or enhanser have increased transcribing of encoding sequence, then it is that operability links to each other with encoding sequence.Used expression vector can adopt various commercially available expression vector well known by persons skilled in the art, for example pET11C bacterial expression plasmid among the present invention.

Then, the expression vector with above-mentioned acquisition transforms proper host cell." host cell " can comprise prokaryotic cell prokaryocyte and eukaryotic cell.In the present invention, preferable host cell is a prokaryotic host cell, for example intestinal bacteria, Bacillus subtilus etc.Term used herein " conversion " is meant that will contain interested expression of nucleic acids carrier with method well known to those skilled in the art directly imports in the host cell.Method for transformation is different because of the host cell type, generally includes: electroporation; Adopt the transfection of calcium chloride, DEAE-dextran or other material; Microparticle bombardment; The fat transfection; Infect and other method (seeing people's such as Sambrook " molecular cloning experiment guide " the 2nd edition, 1989 years).Therefore, the present invention also provides on the other hand through above-mentioned expression vector transformed host cells.

The present invention provides a kind of method for preparing above-mentioned isolated polypeptide on the other hand, it 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); And e) separation and purification obtains described peptide.

A further aspect of the invention provides a kind of vaccine composition, and it contains above-mentioned isolated polypeptide and pharmaceutically acceptable carrier.This vaccine composition can be used for treating tumour and/or human contraception.Term used herein " pharmaceutically acceptable " is meant that they can not produce disadvantageous, hypersensitive or other untoward reaction when molecule body and composition suitably give the animal or human." pharmaceutically acceptable carrier " used herein should be compatible with polypeptide of the present invention, can not reduce the effect of composition with its blend under normal conditions significantly.Suitable carriers normally big, metabolism macromole slowly, as the virion of protein, polysaccharide, poly(lactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer, lipid agglutinator (as oil droplet or liposome) and non-activity.These carriers are selections well known to those of ordinary skill in the art and easy.Vaccine composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, body weight and roughly factor such as disease condition, administering mode determine the useful dosage of patient is used.Vaccine can give together in conjunction with other immunomodulator or immunological adjuvant.

Another one of the present invention aspect provides a kind of isolated polypeptide sequence, and it is by HBsAg ^19-33, HBcAg ^85-140And TT ^580-599Form (92 amino-acid residues of aminoterminal by SEQ ID NO:1 are formed).This carrier can be used for the recombinant multi-epitope chimeric peptide of other target antigens of design construction or the encoding gene of its ().In the coding nucleotide sequence of the former T-cell epitope peptide, TT5 ^80-599Reserved 1 Taq I (TCGA) restriction endonuclease sites in the middle of the epi-position, so a plurality of B-cell epitope of other target antigens tandem gene encode fragment only need its 5 '-end 11 amino acid codings base fragment of synthetic TT epi-position second half section simultaneously, just can finish the full gene fragment of purpose in this Taq I site and splice.

The invention provides that a coding hCG β doubles 6 linear B-cell epitopes (β 5 epitope peptides-β 9 epitope peptides-β 8 epitope peptides-β 5 epitope peptides-β 9 epitope peptides-β 8 epitope peptides) and exogenous 6 Th-cell epitopes (comprise HBsAg ^19-33And TT ^580-599Two broad spectrum epi-positions) synthetic gene (CP22) and recombinant expression plasmid thereof.The pET11c-CP22 recombinant plasmid that makes up can be with inclusion body formal representation chimeric peptide CP22 albumen in intestinal bacteria.Design only contains CP1 and CP10 and the CP7 and the CP11 two class hCG CPs antigen (number of patent applications: 03115893.5 and 03115894.3 of 3 β epi-position half molecular peptides of single group before comparing, these two parts of patent applications are all included this paper in as a reference), the present invention has realized doubling 6 target epitope by genetically engineered and has been connected with the collinearity of T-cell epitope peptide, the CP22 chimeric peptide of expressing has increased the proteic wetting ability of purpose CP22 owing to having deleted each epitope peptide two ends hydrophobic amino acid residues, molecular weight also bigger (being beneficial to preparation property polyacrylamide gel electrophoresis purifying), especially it is better that the animal immune experimental result discloses the CP antigen that only mixes 3 target epi-positions that its antigenicity and immunogenicity compare, promptly only use in the different inbred mouse strains of aluminium salt adjuvant immunity rabbit and can both produce high-level antibody response with 3, and can in the generation antiserum(antisera), detect 3 kinds of anti-each epitope antibodies, compare with the synthetic peptide/DT of reference standard hCG β-CTP, the ability with hCG during body is interior also is significantly improved.Thereby for development immunogenicity hCG biosynthesizing peptide based immunogens better and can produce extensive immunne response in the immune crowd of the different quilt of genetic background is laid a good foundation.The hCG CP22 antigen/encoding gene of institute's unique design can be at expression in escherichia coli, thereby is expected to become possibility for the novel optimization of development and cheapness obtain hCG recombinant multi-epitope CP vaccine reorganization reorganization consistently.

Description of drawings

Fig. 1 is that hCG recombinant multi-epitope chimeric peptide CP22 expresses and the SDS-PAGE of purifying protein analyzes, wherein black arrow indication CP22 expressing protein band.

Fig. 2 is that hCG recombinant multi-epitope chimeric peptide CP22 expresses and the Western blot of purifying protein is identified, wherein M is a protein molecular weight standard, 1 induces CP22 engineering bacteria inclusion body protein sample for 8Mol urea dissolving, 2 do not induce CP22 engineering bacteria inclusion body protein sample for 8Mol urea dissolving, 3 is the CP22 protein sample of purifying, 4 for inducing CP22 engineering bacteria protein sample, and 5 for not inducing CP22 engineering bacteria protein sample.

Fig. 3 is the antibody response level detection behind the CP22 active immunity rabbit.With gathering the average antibody titre that antiserum(antisera) is measured behind purifying CP22 albumen (each 0.5 milligram) the active immunity rabbit (N=6) of aluminium salt adjuvant (Alum) absorption four times on the 7th day.Use hCG β subunit as antigen in the ELESA method, antibody titers is represented with high dilution.Antigen control is respectively the hCG β-CTP/DT of standard and only makes up the CP12 albumen of 3 B-cell epitopes, and the adjuvant contrast is Fu Shi adjuvant (CFA).

Fig. 4 is the antibody response level detection behind the CP22 active immunity mouse.With gathering the average antibody titre that antiserum(antisera) is measured behind purifying CP22 albumen (each 0.1 milligram) three kinds of different inbred lines strain mouse of active immunity (N=4) of Fu Shi adjuvant four times on the 7th day.Use hCG β subunit as antigen in the ELESA method, antibody titers is represented with high dilution.

Fig. 5 gathers the evaluation that each epitope antibodies generates in the antiserum(antisera) behind the CP22 active immunity rabbit, wherein 1 for not expressing the engineering bacteria total protein sample of streptavidin (Stv)-single epi-position (β 5BCE) fusion rotein, 2 for containing the engineering bacteria total protein sample of expressing Stv-β 5BCE fusion rotein, 3 for containing the engineering bacteria total protein sample of expressing Stv-β 9BCE fusion rotein, 4 for containing the engineering bacteria total protein sample of expressing Stv-β 8BCE fusion rotein, and 5 is protein molecular weight standard.

Fig. 6 gathers the evaluation that each epitope antibodies generates in the antiserum(antisera) behind the CP22 active immunity Balb/C mouse, wherein 1 is protein molecular weight standard, 2 for containing the engineering bacteria total protein sample of expressing Stv-β 8BCE fusion rotein, 3 for containing the engineering bacteria total protein sample of expressing Stv-β 9BCE fusion rotein, 4 for containing the engineering bacteria total protein sample of expressing Stv-β 5BCE fusion rotein, and 5 for not expressing the engineering bacteria total protein sample of Stv-β 5BCE fusion rotein.

Fig. 7 gathers the evaluation that each epitope antibodies generates in the antiserum(antisera) behind the CP22 active immunity C3H mouse, wherein 1 is protein molecular weight standard, 2 for containing the engineering bacteria total protein sample of expressing Stv-β 8BCE fusion rotein, 3 for containing the engineering bacteria total protein sample of expressing Stv-β 9BCE fusion rotein, 4 for containing the engineering bacteria total protein sample of expressing Stv-β 5BCE fusion rotein, and 5 for not expressing the engineering bacteria total protein sample of Stv-β 5BCE fusion rotein.

Fig. 8 gathers the evaluation that each epitope antibodies generates in the antiserum(antisera) behind the CP22 active immunity C57 mouse, wherein 1 is protein molecular weight standard, 2 for not expressing the engineering bacteria total protein sample of Stv-β 5BCE fusion rotein, 3 for containing the engineering bacteria total protein sample of expressing Stv-β 5BCE fusion rotein, 4 for containing the engineering bacteria total protein sample of expressing Stv-β 9BCE fusion rotein, and 4 for containing the engineering bacteria total protein sample of expressing Stv-β 8BCE fusion rotein.

Fig. 9 weighs in the experiment in the anti-CP22 antiserum(antisera) of rabbit and the mensuration of hCG performance for the uterus.Inject a hCG for three days on end every day and add the prematurity mouse uterine weight of weighing behind the anti-hCG CP22 of the different extent of dilution rabbits antiserum(antisera), add the anti-standard hCG β of rabbit-CTP/DT vaccine antiserum(antisera) with hCG and add the anti-hCG CP12 of rabbit (only mixing 3 B-cell epitopes) antiserum(antisera) control group with hCG and compare, in the anti-hCG CP22 of the rabbit antiserum(antisera) and the hCG ability obviously improve ( ^▲P＜0.001, ^{▲ ▲}P＜0.005).

Figure 10 A-E has shown that the cell epitope of each hCG chimeric peptide puts in order and aminoacid sequence, and wherein Figure 10 A is that T-and B-cell epitope among the hCG chimeric peptide CP1 puts in order and aminoacid sequence; Figure 10 B has shown that T-and the B-cell epitope among the hCG chimeric peptide CP10 puts in order and aminoacid sequence; Figure 10 C has shown that T-and the B-cell epitope among the hCG chimeric peptide CP7 puts in order and aminoacid sequence; Figure 10 D has shown the T-among the hCG chimeric peptide CP11 and the B-cell epitope puts in order and aminoacid sequence (hCG β 38-57 peptide section contains β 5 epi-positions in above 4 hCGCP chimeric peptide sequences, and hCG β 111-145 peptide section contains β 9 and β 8 epi-positions); Figure 10 E has shown that T-and the B-cell epitope among the hCG chimeric peptide CP22 of the present invention puts in order and aminoacid sequence.

Embodiment

The object of the present invention is to provide coding hCG 3 linear B-cell epitopes of β (doubling) and exogenous 6 Th-cell epitopes (to comprise HBsAg ^19-33And TT ^580-599Two broad spectrum epi-positions) splicing synthetic gene (CP22) and their recombinant expression plasmid.The pET11c-CP22 recombinant plasmid that makes up all can be with inclusion body formal representation chimeric peptide CP22 albumen in intestinal bacteria.The present invention has realized that by genetically engineered hCG β cyclic peptide that immunogenicity is better and the linearity of hCG β-CTP are connected, the hCG chimeric peptide CP22 that expresses since its N end mixed abundant external source broad spectrum or the strong t cell epitope of haplotype and needn't with other high molecular weight protein carrier couplings, thereby hCG biosynthesizing peptide vaccine strong for the development immunogenicity and can produce extensive immunne response in the immune crowd of the different quilt of genetic background is laid a good foundation.The hCG chimeric peptide CP22 (aminoacid sequence is seen SEQ.NO1) of institute's unique design and encoding gene (the DNA base sequence is seen SEQ.NO2) thereof can be at expression in escherichia coli, and the animal immune experimental result proves: the CP22 multi-epitope chimeric peptide antigen is not with the carrier coupling of external source high molecular weight protein and only also can produce strong immune response in being tried rabbit and mouse with aluminium salt adjuvant; This antigen can produce same antibody response in 3 kinds of different inbred lines strain mouse; Can both detect three kinds of hCG β linear epitope antibody in the anti-designed molecule in rabbit and the mouse anti CP22 antiserum(antisera) respectively; Compare with the hCG β-CTP/DT chemosynthesis peptide vaccine of standard, the performance with natural hCG in the anti-hCG CP22 antiserum(antisera) obviously strengthens (remarkable statistical significance is arranged).Therefore, hCG CP22 recombinant multi-epitope chimeric peptide might become the neoantigen of development human contraception and oncotherapy.

According to immunology principle and hCG vaccine research present situation, apply the major obstacle that is faced at it especially, for example, how can cheapness obtain the hCG vaccine antigen consistently? how on the specific basis of tool, to increase in the synthetic peptide antiserum(antisera) of hCG β-CTP and the performance of hCG? can avoid using high molecular weight protein carrier and special adjuvant that the required Th cell epitope of immunne response in the body is provided, to make things convenient for vaccine preparation production and to reduce production costs? in addition, the problem that also has " vaccine One Hundred Family Names ", promptly how to avoid MHC heredity restriction, the effective immunne response rate that comprises hCG hormonal dependent malignant tumor patient desire contraception women's the immune crowd of quilt? we have designed the hCG chimeric peptide immunogen CP22 of the novel optimization that is intended to overcome the above problems, and it is by 3 linear ratio cell epitopes (doubling) among the hCG β and comprise that 6 external source Th cell epitopes of 2 broad spectrum epi-positions form.

The CP22 multi-epitope chimeric peptide that the invention provides unique design splices and combines sequence, i.e. HBsAg ^19-33-HBcAg ^85-140-TT ^580-599-βZhuan Jiao peptide-hCG β ^42-54Wetting ability fragment-hCG β among the-hCG β ^109-122-hCG β ^132-145Wetting ability fragment-hCG β among the-hCG β ^42-54-hCG β ^111-121-hCG β ^133-140Each epi-position fragment of CP22 among the SEQ.NO1, except that βZhuan Jiao peptide (Leu-Ser-Pro-Gly), all select oneself listing clinical application or unusually clinical trial do not have the vaccine component of safety problem.The epitope peptide of hCG β and wetting ability fragment coding gene order are according to hCG β cDNA sequence (the Fiddes JC and Goodman HM.The cDNAfor the β-subunit of human chorionic gonadotropin suggests evolution of a gene byreadthrough into the 3 ' untranslated region.Nature 286:684-687 that has cloned, 1980), wherein the partial amino-acid codon has been used the codon of intestinal bacteria preferences instead.

β 5 in the HCG CP22 chimeric peptide ^45-52Antibody neutralizing epitope and β 9 ^113-116With β 8 ^137-144The following document of publishing of selection foundation of two specific epitopes: Stevens VC, et al.The identification of peptidesequences of human chorionic gonadotropin containing a conformational epitope.ImmunolLitters 12:11-18,1986; Dirnhofer S, et al.The molecular basis for epitopes on the free β-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide andthe hCG β-core fragment.J Endocrinol 141:153-162,1994.Consider that biosynthetic peptide needs certain length, and make things convenient for expression product to be convenient to the SDS-PAGE electrophoresis detection, and suitably increase synthetic peptide length and also help immunogenicity and strengthen this some, in purpose CP molecular designing, used the relative hydrophilic amino-acid residue in band two ends.In addition, for the wetting ability that strengthens molecules of interest and/or improve the protein molecular conformation, wetting ability fragment (8 or 6 residues, indivedual residues have been done adjustment) and βZhuan Jiao peptide among the hCG β in the CP22 molecule, have been mixed respectively.

HCG CP22 chimeric peptide of the present invention is by above-mentioned each epi-position or peptide fragment encoding gene fragment assembly, and add Ecor I-Nde I restriction enzyme site and ATG initiator codon at its 5 ' end, and after its 3 ' end end connects TAA terminator codon and BamH I restriction enzyme site, reading with complete purpose chimeric peptide that the frame reorganization inserts can be through this bacterial expression plasmid of IPTG inductive pET11c, has finally realized the expression of CP22 in low-cost and the intestinal bacteria being convenient to operate.Expression product is through with discerning hCG β ^133-139The immunoblotting of the monoclonal antibody OT3A of sequence (Western blotting test) is identified, has been confirmed that the hCG CP22 chimeric peptide of designed structure accounts for the specifically expressing of total protein of cell about 1%.

In the recombinant multi-epitope hCG CP22 that the present invention makes up, the selection of six cell epitopes of its N end is according to following discloses information:

Widely known to, HBsAg one can bring out the powerful immunogen that autoantibody generates, and shows that itself has strong Th-cell epitope.The HBsAg that is identified ^19-33Then be can by different HLA-DR and-" broad spectrum " t cell epitope of DQ haplotype identification.Reports such as Greenstein JL: this peptide and HIV-1 tunicle gp120 the 3rd variable region structural domain (V3 ring, the effective neutralizing antibody of energy guiding district) chimeric peptide of collinearity chemosynthesis, only just in whole 6 kinds of different inbred mouse strains, all bring out and produced anti-V3 cyclic peptide antibody (A universal T cellepitope-containing peptide from hepatitis B surface antigen can enhance antibody specificfor HIV gp120.J Immunol 148:3970-3977,1992) with the absorption of aluminium salt adjuvant;

2. in the vaccine of using that goes on the market, HBcAg also is a kind of powerful immunogen.Each t cell epitope that exists in its 85-140 peptide is also identified (Tiollais P, et al.Biology of hepatitis B virus.Science, 1981 already; 213:406-411; Milich DR, et al.Hepatitis B synthetic immunogen comprised ofnucleocapsid T-cell sites and an envelope B-cell epitope.Proc Natl Acad Sci USA.1988; 85:1610-1614).Now known HBcAg ^85-100Be H-2 ^d, HBcAg ^100-120Be H-2 ^fAnd H-2 ^q, HBcAg ^120-131Be B10.S (H-2 ^s), HBcAg ^129-140Be B10 (H-2 ^b), and HBcAg ^120-140Be H-2 ^{S, b}The complementary cell epitope of the restricted T of MHC II class.Very clear, HBcAg ^85-140At least contain four Th-cell epitopes in the peptide.Select this section of HbcAg peptide of forming by 57 amino-acid residues for use, be for improving the T cytositimulation broad spectrum of constructed chimeric peptide, thereby reaching the molecule adjuvant effect that strengthens this t cell epitope peptide section, is the consideration that needs certain-length for the biosynthesizing peptide too;

3. well-known, the definition of wide spectrum is comparatively speaking.Therefore, in order in the immune crowd of genetic background divergence, to produce immunne response rate as much as possible near 100%, in the purpose chimeric peptide of our place design again and broad spectrum Th-the cell epitope---TT that has used another also widely to know and use ^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 the hCG multi-epitope chimeric peptide action thing immunology detection of going forward side by side, its

Concrete steps are as follows:

(1) design of hCG CP22 chimeric peptide total length code cDNA, each encoding gene fragment is chained together in order, 5 '-end adds initiator codon ATG and EcoR I-Nde I viscosity restriction enzyme site base before, on 3 '-termination two terminator codon TAATGA with and subsequent BamH I viscosity restriction enzyme site base, form complete CP22 encoding gene (SEQ.NO2);

(2) the positive minus strand of CP22 encoding gene is respectively resolved into 12 fragments and carry out chemosynthesis, by the DNA recombinant technology they are spliced afterwards;

(3) clone of hCG CP22 chimeric peptide total length code cDNA and order-checking are identified;

(4) structure of pET11c-hCG recombinant expression vector and the expression in intestinal bacteria thereof, expression vector is selected the pET11c plasmid for use, enzyme from the pBS/CP22 plasmid of identifying through dna sequencing cuts out the CP22 gene with Nde I and two kinds of restriction enzymes of BamH I, and the pET11c plasmid is inserted in reorganization then.This recombinant expression plasmid is identified through dna sequencing once more, is transformed BL21 (DE3) plysS host bacterium then respectively;

(5) separation and purification of purpose expression product CP22;

(6) active immunity rabbit and different inbred lines strain mouse, the check antigenicity of CP22 and immunogenicity with and cross in MHC heredity restriction and its antiserum(antisera) and the performance of hCG.

Reorganization hCG multi-epitope chimeric peptide CP22 provided by the invention has made up in the target antigen hCG β subunit whole 3 linear B-cell epitopes, and (totally 6 of doubles, wherein second β 8 epi-position is with OT3A monoclonal antibody recognition sequence ^133-139Substitute) and extrinsic protein comprise 6 Th-cell epitopes of two broad spectrum epi-positions.Because recombinant expressed hCGCP22 immunogen had both been possessed β 9 and 8 two specific B-cell epitopes of β of hCG β-CTP chemical synthesising peptide, β 5 these antibody neutralizing epitopes have also been mixed simultaneously, therefore in the animal antiserum when detecting its specific anti-β 9 of reflection and β 8 antibody, also obviously improved in it and the ability of hCG because of the existence of β 5 antibody.These related experiment evidences show that the present invention can form new hCG immunotherapy of tumors and/or human immunity contraception synthetic peptide vaccine preparation method and biological products.6 T complementary (Th-) cell epitope polyphone peptide half point among the CP22 has been selected 1 T-cell epitope HBsAg in the hepatitis B surface antigen for use ^19-33, comprise the peptide section HBcAg of 4 T-cell epitopes in the hepatitis B virus core antigen ^85-140With 1 T-cell epitope TT in the Toxoid,tetanus ^580-599The T-cell epitope peptide of series combination can work the molecule adjuvant effect of transferring t cell response secretion Th2 cytokines in the vivo immuning system, thereby such CP immunogen no longer needs with the external source high molecular weight protein coupling that the Th-cell epitope is provided and uses special immunological adjuvant, makes things convenient for vaccine preparation production (can get final product with the absorption of aluminium salt adjuvant through the people) simultaneously.HBsAg wherein ^19-33And TT ^580-599Two epitopic features are that they all are the T-cell epitopes of strong and broad spectrum.Both combination and usefulness in 1 recombinant multi-epitope chimeric peptide, help to overcome ubiquitous in the vaccine development " vaccine One Hundred Family Names " this difficult problem, both might (major histocompatibilitycomplex, MHC) the genetic background difference have been produced to greatest extent the effective immunne response rate near 100% among the immune crowd in main histocompatibility complex by them.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, carry out all according to the condition described in " molecular cloning: laboratory manual " (third edition) (Cold Spring HarborLaboratory Press) of people such as normal condition such as Sambrook, or according to the condition that manufacturer advises.

Embodiment

Material and method

1. restriction enzyme EcoR I, Sal I, Nde I, BamH I is a U.S. Boehringer Mannheim company product, the T4DNA ligase enzyme is a Britain Biolabs product, the T4 polynucleotide kinase is a U.S. New EnglandBiolabs product, Taq I archaeal dna polymerase is genetic engineering National Key Laboratory of a Fudan University product, IPTG is a U.S. Promega product, protein lower molecular weight standard and alkaline phosphatase connection sheep anti mouse two anti-(IgG/HRP) are Huamei Bio-Engrg Co.,'s product, 5-bromo-4-chloro-3-indoles-β-D galactoside (X-gal) and isopropylthio-β-D galactoside (IPTG) are U.S. Sigma company product, identification hCG β ^133-139The monoclonal antibody OT3A of sequence is so kind as to give by Dutch Organon Technika.

2. clone and sequencing vector pBluescript KS (PBS) are available from Stratagene company, and expression vector pET11c is a Novagen company product.E. coli tg1 and BL21 (DE3) are genetic engineering National Key Laboratory of Fudan University preservation strain.

3.QIAprep it is German QIA company product that SPIN plasmid extraction test kit, QIAquick PCR purification kit and QIAquick glue reclaim test kit.

4. 24 (12 pairs) nucleotide base fragments of the positive minus strand of designed CP22 encoding gene are given birth to worker company by Shanghai and are synthesized.

5. laboratory animal: (body weight 2.0 ± 0.5kg) is provided by SIPPR-BK Lab Animal LtdCo. (Shanghai) new zealand male rabbit.Balb/C (the H-2 in 6-9 week ^b), C3H/hej (H-2 ^k) and DBA/1 (H-2 ^q) female mice (18-20g) is respectively available from SIPPR-BK Lab Animal Ltd Co. (Shanghai) and Chinese Academy of Sciences's Shanghai Experimental Animal Center.HCG, hCG β subunit and Fu Shi adjuvant are available from U.S. Sigma company, and aluminium salt adjuvant is available from Shanghai Vaccine and Serum Institute.Standard WHO hCG β-CTP/DT synthetic peptide vaccine is so kind as to give by professor Stevens of Ohio State Univ-Columbus USA.

The concrete steps of preparation are as follows:

1, the design of the complete encoding gene of hCG CP22

Foreign protein T-among the hCG chimeric peptide CP22 and target antigen double B-cell epitope and other arrangement of fragments amino-acid sequence sequences are seen SEQ ID NO:1.

The design of hCG CP22 full-length cDNA is finished under area of computer aided, reads the codon of selecting the intestinal bacteria preference in the frame substantially for use, splices design by the encoding gene fragment that each T-cell epitope peptide section and B-cell epitope peptide section have been published.Through the PC-GENE software retrieval, the composition base of indivedual codons is adjusted, to eliminate possible positive and negative tumor-necrosis factor glycoproteins, comprise that base complementrity situation in overlap occurs and inappropriate restriction enzyme site between fragment and the fragment.HCG CP1 gene read 5 of frame '-end increased initiator codon ATG and before EcoR I-Nde I viscosity restriction enzyme site, in its 3 '-end added two terminator codon TGATAA with and subsequent BamH

I viscosity restriction enzyme site.

2, the splicing of hCG CP22 complete genome

The CP22 gene of 562 base pairs of total length (positive minus strand two ends reserved EcoR I and BamH I sticky end) is divided into 24 and poly-ly synthesizes to the 57 poly-oligonucleotide fragments that are uneven in length from 31.Add ddH after synthetic ₂O is diluted to each fragment of 20pmol/ μ l and identifies purity through 16% polyacrylamide sex change gel electrophoresis earlier, respectively get then 16 μ l (do not comprise positive minus strand 5 '-two fragments of end) under 0.5 μ l 10U/ μ ld T4 polynucleotide kinase (adding 2 μ l10mmol/L ATP) catalysis, carry out 18 segmental 5 '-the end phosphorylated.That continues makes the positive minus strand homologous segment renaturation of annealing in twos respectively, allows adjacent dna fragmentation classification link to each other in twos by enzymatic reaction again.Last complete genome ligation liquid separates through 5% native polyacrylamide gel electrophoresis, ethidium bromide (EB) dyeing, and total length target gene fragment under the contrast molecular weight standard cuts under long wavelength ultraviolet light then reclaims DNA with " crushing infusion method ".

3, the clone of hCG CP22 gene and order-checking

The CP22 gene fragment of reserving the EcoR I at gene two ends and BamH I sticky end is directly inserted also pBS cloning and sequencing carrier through EcoR I and BamH I double digestion by the DNA reorganization, use enzyme ligation liquid transformed into escherichia coli TG1 host bacterium again, at last screening may be the white colony of recombinant clone on the LB culture medium flat plate of the IPTG of the X-gal of coating 20% and 100mmol/L.Some extractive recombinant plasmids are done the analysis of gene base sequence on the PAGE glue of ABI373A type automatic dna sequencer after preliminary enzyme is cut evaluation.The final positive colony of determining that desired insertion gene order is consistent with design.

4, the structure of pET11c/hCG CP22 recombinant expression vector and the expression in intestinal bacteria thereof

Expression vector has been selected Tong Guo the IPTG inductive pET11c plasmid that has strong T7 promotor for use.Enzyme from the pBS/CP1 plasmid of identifying through dna sequencing cuts out the CP22 gene fragment with NdeI and two kinds of restriction enzymes of BamH earlier, then the Nde I and the BamH I site of the pET11c plasmid is inserted in its reorganization with the T4DNA ligase enzyme.This recombinant plasmid is difference transforming protein deficient host bacterium BL21 (DE3) plysS after dna sequencing is identified once more.The abduction delivering condition of engineering bacteria BL21 (DE3)/pET11c-CP22 is as follows: inoculate the purpose engineering bacteria in adding contains the 50ml LB substratum (250ml shake bottle) of 50 μ g/ml ampicillin and 34 μ g/ml chloramphenicol.37 ℃ jolt the OD that cultivation made culture in 3-4 hour ₆₀₀Value reaches 0.5, gets the 3ml starting culture and is inoculated in the 500ml LB substratum (2000ml shakes bottle) that contains 500 μ g ampicillin and 340 μ g chloramphenicol, and 37 ℃ jolt the OD that fermentation made culture in 3-4 hour ₆₀₀Value reaches 0.8-1.0, adds IPTG (final concentration 1.0mM) then and induces, and continues fermentation 4-5 hour.Bacterium liquid 4 ℃ following 5000 rev/mins centrifugal 15 minutes, collect bacterial precipitation.

5, the SDS-PAGE of purpose expression product analyzes and Western blot (Western blot) evaluation.

Bacterial precipitation thing through abduction delivering is resuspended in the bacterial lysate with the ratio that every gram wet thallus adds 5ml solution, ultrasonication bacterium 0.5-1 hour, in 4 ℃ of centrifugal collection bacterial sediments, add again lysate that 50ml contains 0.5%Triton carry out ultrasonic beat even, 4 ℃ of centrifuging and taking precipitate afterwards, add 50ml lmol/L urea and wash, after centrifugal sediment added 25ml 8mol/L urea ultrasound suspending, the centrifuging and taking supernatant carried out SDS-PAGE and analyzes.Second half gel that repeats sample carries out electric transfer printing, the purpose expressing protein on the nitrocellulose membrane by with the anti-reaction of the sheep anti mouse two of special monoclonal antibody OT3A (is anti-) and horseradish peroxidation alkali phosphatase enzyme mark after, develop the color with DAB liquid.

6, the separation and purification of purpose expression product CP22

With the preparation SDS-PAGE method [Zou Yongshui, Xu Wanxiang etc.: the polyacrylamide gel electrophoresis of R-HCG's chimeric peptide 12 preparation. Acta Biochimica et Biophysica Sinica, 2002; 34 (5): 671-674] single step purification CP22 expressing protein, every liter of intestinal bacteria nutrient solution can obtain the target protein that the electrophoretic band homogeneity is higher than the 0.1-0.5 milligram more than 95%.Lipidated protein detects by the SDS-PAGE method.

7, use the immunology detection of hCG CP22 purifying protein active immunity animal

(1) New Zealand white rabbit (N=6) active immunity: active immunity adopts back multi-point injection method.It is fully emulsified or grind absorption, each every injection 1ml after the 0.5mg of 0.5ml physiological saline solution CP22 albumen adds 0.5ml Fu Shi Freund's complete adjuvant (CFA) with 1ml aluminium salt adjuvant.The incomplete Fu Shi adjuvant of injection 0.5ml (IFA) emulsive 0.5mg CP immunogen after 10 days.Every other week more respectively at rabbit front foot palm intramuscular booster immunization secondary.Reference standard hCG

β-CTP/DT vaccine is only used the CFA adjuvant.Control group is only injected CFA or aluminium salt adjuvant in addition.

The active immunity of (2) three kinds of tool difference-2 haplotype inbred lines strain mouse: the 0.1mg CP22 proteantigen that the 1st week is subcutaneous at the mouse buttocks and the neck multi-point injection adsorbs with the grinding of 0.1ml CFA adjuvant, carry out immunity with 0.1ml IFA emulsification through the CP22 of 0.5ml physiological saline solution immunogen after 10 days once more.Every other week more respectively at mouse front foot palm intramuscular booster immunization secondary.

(3) the ELESA antibody titer is measured: the mensuration of antibody titer adopts the ELESA method.Behind the 4th booster immunization, extract rabbit ear edge venous blood every 1 week, detect its serum antibody titer.The 3rd week after the immunity first time takes a blood sample to mouse tail vein, and afterwards weekly/take a blood sample once in per two weeks, measure its serum antibody titer.HCG β subunit with U.S. Sigma company in the ELESA method is antigen coated enzyme plate, and the antiserum(antisera) that adds serial dilution is one anti-, and goat-anti rabbit/mouse IgG-HRP is two anti-, H ₂O ₂The colour developing of-OPD system is at last at ELX 800

(Bio-TEK Instruments Inc.) measures Universal Microplate Reader.

(4) each BCE antibody generates and detects in the immune serum: before we made up respectively can be in intestinal bacteria β 5, the β 9 of high expression level and β 8 single epi-positions the streptavidin fusion rotein [Xu Wanxiang etc.: the single B-cell epitope of human chorion gonadotrophic hormone beta subunit (β 5, β 9 or β 8) Expression of Fusion Protein and purifying. biotechnology journal, 2004; 20 (1): 49-53].With their derivative engineering bacteria total proteins is that sample (antigen) is walked SDS-PAGE, then with the expressing protein electrotransfer to nylon membrane, back with the of short duration dyeing of ponceau at purpose expressed fusion protein punching two ends, washing decolouring again is an anti-western blot analysis that carries out with the rabbit/mouse resisting anteserum of high titre at last.

(5) the Mouse Uterus experiment of weighing: select 18-25 days Balb/C female mice for use, hCG standard substance (available from U.S. Sigma company) with 200ng, make it respectively to carry out intraperitoneal injection for three days on end with dilution antiserum(antisera) with the rabbit anti-serum stoste of CP12, CP22 and contrast β-hCG CTP: DT, 1: 10 and 1: 50, last is injected execution in back 24 hours, the mouse uterine weight of weighing.Feminine gender and positive controls be injecting normal saline and hCG respectively.

Result of study shows, the present invention has successfully been spliced synthetic and has been made up hCG chimeric peptide CP22 artificial gene and pET11c/CP22 recombinant expression plasmid, can go out the CP22 target protein with the inclusion body formal representation after transforming host e. coli BL21 (DE3) plysS with them, and obtain checking (Fig. 1 and Fig. 2) by protein blot experiment.

The SDS-PAGE analytical results shows that the expression level of hCG chimeric peptide CP22 in intestinal bacteria is about 1%, can gather in the crops 0.5mg electrophoresis homogeneity by every liter of engineering bacteria nutrient solution of preparation SDS-PAGE method and be higher than 90% target protein (Fig. 1 and Fig. 2).

The animal immunology experiment has also obtained expected results: 1, because designed CP22 chimeric peptide has mixed 6 external source T-cell epitopes, only choose available aluminium salt adjuvant active immunity rabbit and mouse, CP22 can produce and use the very approaching antibody response level (Fig. 3 and Fig. 4) of Fu Shi Freund's complete adjuvant result; 2, in rabbit and mouse anti CP22 antiserum(antisera), can both detect β 5, β 9 and 8 three kinds of antibody of β of being mixed and have (Fig. 5～Fig. 8), show that each epi-position is not offered in the immunity system antigen processing treatment in vivo in the synthetic peptide of CP22 with being damaged; 3, owing to selected two broad spectrum T-cell epitopes for use in the T-cell epitope peptide, in by three kinds of different inbred lines strain mouse of immunity, all produced the very approaching antibody response of level, comprise and have each epitope antibodies (Fig. 4, Fig. 6～Fig. 8), prompting CP22 antigen tentatively has the performance of crossing over MHC heredity restriction; 4, owing in CP22 recombinant multi-epitope chimeric peptide, mixed β 5 antibody neutralizing epitopes, with only contain the standard β-hCG CTP of β 9 with 8 two specific B-cell epitopes of β: DT chemosynthesis peptide vaccine is compared, in its body in and the ability of hCG obtain to have the raising (table 1 and Fig. 9) of statistical significance.

Table 1 uterus is weighed in the experiment in the anti-CP22 antiserum(antisera) of rabbit and the mensuration of hCG performance

Group	Uterus weight (milligram/gram body weight)
				Extent of dilution 1: 1	1∶10	1∶50
	Saline control hCG contrast hCG+CP12 antiserum(antisera) hCG+CP22 antiserum(antisera) hCG+CTP antiserum(antisera)	9.90±0.62 28.88±1.41 12.30±0.37 ▲ 10.04±0.29 ▲ 15.16±0.90 ▲▲	9.90±0.62 28.88±1.41 14.00±0.24 ▲ 11.48±0.27 ▲ 16.68±0.53 ▲▲				9.90±0.62 28.88±1.41 15.46±0.43 ▲▲ 14.46±0.91 ▲ 19.12±0.87 ▲▲

Obviously, the structure of hCG chimeric peptide CP22 mosaic gene and the acquisition of expressing protein thereof, the good animal immunology detected result of particularly showing, the novel optimization hCG synthetic peptide vaccine that is used for hCG hormonal dependent treating malignant tumor and human fertility goal of regulation and control for development is laid a good foundation, and other are antiviral and/or parasite synthetic peptide vaccine field is also significant in development.

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.

Sequence table

＜110〉Shanghai Family Planning Science and Research Institute.

Fudan University

＜120〉antigenic molecular designing of human chorionic gonadotrophin recombinant multi-epitope chimeric peptide CP22 and preparation method thereof

<130>052542

<160>2

<170>PatentIn?version?3.1

<210>1

<211>183

<212>PRT

<213>1

<400>1

Met?Phe?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Pro?Gln?Ser?Leu?Asp

1 5 10 15

Val?Val?Ser?Tyr?Val?Asn?Thr?Asn?Met?Gly?Leu?Lys?Phe?Arg?Gln?Leu

20 25 30

Leu?Trp?Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu

35 40 45

Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr

50 55 60

Arg?Pro?Pro?Asn?Ala?Pro?Ile?Leu?Asn?Ser?Val?Asp?Asp?Ala?Leu?Ile

65 70 75 80

Asn?Ser?Thr?Lys?Ile?Tyr?Ser?Tyr?Phe?Pro?Ser?Val?Gly?Pro?Ser?Leu

85 90 95

Thr?Arg?Val?Leu?Gln?Gly?Val?Leu?Pro?Ala?Leu?Pro?Gln?Gly?Thr?Thr

100 105 110

Thr?Asp?Tyr?Gly?Gly?Thr?Cys?Asp?Asp?Pro?Arg?Phe?Gln?Asp?Ser?Ser

115 120 125

Ser?Ser?Lys?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro

130 135 140

Gln?Thr?Thr?Asp?Tyr?Gly?Ser?Thr?Arg?Val?Leu?Gln?Gly?Val?Leu?Pro

145 150 155 160

Ala?Leu?Pro?Gln?Asp?Asp?Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Arg

165 170 175

Leu?Pro?Gly?Pro?Ser?Asp?Thr

180

<210>2

<211>549

<212>DNA

<213>1

<400>2

atgtttttcc?tgctgacacg?catcctgaca?atcccgcagt?ctctggacgt?agtatcttat 60

gttaatacca?acatgggtct?gaagttccgt?caactgcttt?ggtttcatat?ctcttgcctt 120

acatttggcc?gtgagacagt?acttgaatat?ctggtatctt?tcggtgtatg?gattcgcacc 180

ccgcctgcgt?atcgtcctcc?gaatgctcct?atccttaatt?ctgttgatga?cgcactgatc 240

aattcgacca?aaatttattc?atattttccg?tctgtaggtc?cgtctctgac?ccgcgttctg 300

cagggtgttc?tgccggccct?gcctcagggt?acgacaaccg?actacggtgg?tacctgtgat 360

gatccgcgct?tgcaagattc?ctcaagttca?aagtcacgtc?tgccgggtcc?ctcagacacc 420

ccgatcctgc?ctcaaaccac?tgactacgga?tccacccgcg?ttcttcaggg?tgttctgccg 480

gccctgcctc?aggatgatcc?gcgcttccag?gactcttcct?cctcccgtct?gccgggtccg 540

tcagacacc 549

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.

Images