CN1562363A - Polypeptide vaccine of free from adjuvant for anti human chorionic gonadotrophin and preparation method - Google Patents
Polypeptide vaccine of free from adjuvant for anti human chorionic gonadotrophin and preparation method Download PDFInfo
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Abstract
A human choriogonadotropin plypeptide vaccine without adjuvant for preventing and treating breast cancer, colon cancer, pancreas cancer and prostate cancer, or for contraception isp repared through repeatedly inserting the two repeated antigen epitope polypeptide fragments serially connected gene into the downstream of the heat shock protein HSP65 gene coming from mycobacterium bovis to realize the fusion expression of them.
Description
Technical field
At the tumor area of medical domain, can use tumor vaccine usually, technology of the present invention is a kind of polypeptide vaccine of anti-hCG, can be used for the treatment of the human chorionic gonadotropin dependent tumors.
Background technology
Human chorionic gonadotropin (Human Chorionic Gonadotropin, HCG) be by the excretory a kind of glycoprotein hormones of people's embryo trophoblastic cell, its effect is to keep and stimulate corpus luteum of pregnancy to produce progesterone and estradiol, promote endometrial cell to reach maturity, be the embryo nidation preparatory condition, have important effect in early days in the gestation.HCG is made up of α, β two subunits, and the α subunit of α subunit and human interstital-cell-stimulating hormone, follicle stimulating hormone and thyrotropin is basic identical, and the specificity of hormone, activity are the β subunit.In addition, the tumor-selective secretion strand β HCG of multiple tissue or the core fragment of β HCG, as the choriocarcinoma in the reproductive system, carcinoma of prostate, breast carcinoma, carcinoma of endometrium, cervical cancer, as gastric cancer, bladder cancer, pulmonary carcinoma, hepatocarcinoma, even the squamous cell carcinoma in the oral cavity also can detect in the other system.HCG by with target cell on receptors bind transmit hormone information, the HCG receptor is widely distributed, does not exist only in the gonad target tissue, and as prostate, mammary gland, adrenal gland, thyroid, cerebral cortex etc. expression is arranged also in some non-gonadal tissues.Found in recent years that the LH/HCG receptor also was present in some malignant cell surface, as carcinoma of endometrium, cervical cancer, ovarian cancer, gastric cancer etc.Therefore, β HCG also is one of effective target molecule of anti-curing cancers as one of characteristic molecule of tumor cell secretion.Many experiments have proved that the vaccine that utilizes β HCG purifying protein, recombiant protein and synthetic polypeptide to make can excite generation protection antibody in the body; in and the β HCG antigen of tumor cell secretion; and by the combining of blocking-up HCG and tumor cell surface HCG specific binding site, realization is to tumor treatment.In addition, thus can suppress fetal development by blocking-up HCG causes miscarriage performance contraceptive efficacy.
The foreign scholar holds 37 peptides and diphtheria toxin, diphtherotoxin or tetanus toxin coupled with the C of β HCG, induces body to produce anti-HCG antibody with this coupled thing, and demonstration can be used for the treatment of colon cancer, cancer of pancreas and breast carcinoma etc.In China, diphtheria toxin, diphtherotoxin or tetanus toxin all have been listed in the planned immunization vaccine, and many people were once used diphtheria toxin, diphtherotoxin and tetanus toxin immunity mistake.When using diphtheria toxin, diphtherotoxin or the coupled thing of tetanus toxin in these crowds, immune effect can weaken.Do not have the similar epi-position of diphtheria toxin, diphtherotoxin or the coupled thing of tetanus toxin to suppress problem with polypeptide vaccine, but immunogenicity how to strengthen polypeptide vaccine is the target that many vaccine research personnel pursue.
Usually, polypeptide vaccine need add adjuvant, could produce enough strong immunne response by excitating organism.Many adjuvants commonly used only can be used for animal as Fu Shi Freund's complete adjuvant and freund 's incomplete adjuvant etc., can not be used for human body; The available adjuvant of human body only has aluminium adjuvant at present, and this adjuvant usually can not play the effect of reinforced immunological originality to polypeptide vaccine.
Summary of the invention
The purpose of this invention is to provide a kind of method of strengthening immunogenicity of polypeptides.
Another object of the present invention provides a kind of anti-HCG polypeptide vaccine of adjuvant-free.
A further object of the present invention provides the method for producing the anti-HCG polypeptide vaccine of this adjuvant-free.
Purposes that purpose is the anti-HCG polypeptide vaccine of adjuvant-free in addition of the present invention.
In a first aspect of the present invention, a kind of method of strengthening immunogenicity of polypeptides is provided, this method is the downstream that two sections multiple antigen epitope polypeptide tandem genes are inserted the HSP65 gene repeatedly, makes multiple antigen epitope polypeptide and the HSP65 amalgamation and expression of connecting again.The principle of this reinforcement immunogenicity of polypeptides method is: (1). antigen epitope polypeptide is repeated repeatedly, can increase the dosage of epitope, increase whole antigenic molecular weight, thereby increase the immunogenicity of antigen epitope polypeptide; (2). realize the repeatedly repetition of epitope by the clone of circulation repeatedly who repeats antigen epitope genes; (3). mycobacteria (bacillus calmette-guerin vaccine) HSP65 has the function of adjuvant on the throne, and the albumen that antigen epitope polypeptide is repeated segment and mycobacteria (bacillus calmette-guerin vaccine) HSP65 amalgamation and expression can not need add adjuvant as vaccine, brings out body and produces intensive immunne response.And, HSP65 and epitope amalgamation and expression can be saved the step of antigen epitope polypeptide and adjuvant HSP65 on the throne chemical coupling.
In the present invention, term " X
2n" refer to epitope arranged in series, X
2Refer to two sections β HCG109-118 tandem polypeptides, n refers to be used for the repetition epitope (X of arranged in series
2) number, can equal 1 or greater than 1, can directly link between the epitope, also can spaced amino acid residue.In vaccine research, can be spatially adjacent between the epi-position also can be non-conterminous.
In a second aspect of the present invention, provide a kind of anti-hCG adjuvant-free polypeptide vaccine.This vaccine adopts the described method of a first aspect of the present invention to design.In the vaccine molecule, (repetition epitope) n equals ThrCysAspAspProArgPheGlnAspSerThrCysAspAspProArgPheGlnAs pSerAlaArgThrCysAspAspProArgPheGlnAspSerThrCysAspAspProA rgPheGlnAspSerAlaArgThrCysAspAspProArgPheGlnAspSerThrCys AspAspProArgPheGlnAspSerAlaSer, promptly contains six sections (but being not limited to) β HCG109-118 peptide section as epitope; In order to narrate conveniently, in an embodiment, the present invention contains the epitope " X of six sections β HCG109-118
6" expression.β HCGCTP37 is meant HCG β subunit 10 9-145 polypeptide, be that HCG β chain C holds 37 peptides, its aminoacid sequence is ThrCysAspAspProArgPheGlnAspSerSerSerSerLysAlaProProProSe rLeuProSerProSerArgLeuProGlyProSerAspThrProIleLeuProGln.HSP65 is meant the heatshock protein that comes from as the mycobacteria source of bacillus calmette-guerin vaccine, and international gene bank accession number is M17705.With antigen epitope polypeptide and HSP65 amalgamation and expression, can strengthen the immunogenicity of polypeptide vaccine, need not add adjuvant and just can excitating organism generation strong immunization reply.The adjuvant-free anti-hCG polypeptide vaccine that β HCGCTP37 and six sections epitopes and HSP65 amalgamation and expression are obtained is expressed as " HSP65-X
6-β HCGCTP37 ".
In a third aspect of the present invention, provide the method for the repetition epitope polypeptide vaccine of producing anti-hCG, details are as follows for its technology path:
1. the design of β HCGCTP37 gene and acquisition
Under the prerequisite that does not change β HCGCTP37 polypeptid acid sequence, select the codon of escherichia coli preference for use, by means of four oligonucleotide of Computer Design, obtain the nucleotide sequence of β HCGCTP37 polypeptide gene by the PCR method, the recognition site that 5 ' end has added BamHI, the recognition site that 3 ' end has added termination codon and HindIII.
2.HSP65 the design of gene and acquisition
Under the prerequisite that does not change the HSP65 aminoacid sequence, select the codon of escherichia coli preference for use, by means of two oligonucleotide of Computer Design, from the DNA that commercially available bacillus calmette-guerin vaccine extracts, obtain the nucleotide sequence of HSP65 gene by the PCR method, the recognition site that 5 ' end has added NcoI, the recognition site that 3 ' end has added NheI and BglII.
3. repeat epitope (X
2) design and the acquisition of gene
Under the prerequisite that does not change two sections β HCG109-118 tandem polypeptide aminoacid sequences, select the codon of escherichia coli preference for use, by means of two oligonucleotide of Computer Design, obtain to repeat the nucleotide sequence of antigen epitope polypeptide gene by the PCR method, the recognition site that 5 ' end has added XbaI, the recognition site that 3 ' end has added NheI.The important point is that the codon of one of them serine Ser is selected TCA for use, so that the screening of positive colony.Because vector plasmid adopts single endonuclease digestion, insert X
2Have forward and reverse two kinds of situations during gene, TCA is expressed as Ser if forward inserts then, HSP65-X
2nThe proteic molecular weight ratio HSP65-X of-β HCGCTP37
2 (n-1)It is big by about 2400 that-β HCGCTP37 albumen is wanted; TCA reverse sequence TGA is a termination codon if oppositely insert then, HSP65-X
2nThe proteic molecular weight ratio HSP65-X of-β HCGCTP37
2 (n-1)The proteic molecular weight of-β HCGCTP37 is little.So can judge that with the SDS-PAGE coomassie brilliant blue staining forward correctly inserts X
2The recombinant clone of gene.
4.pED-the structure of β HCGCTP37 recombination engineering bacteria
β HCGCTP37 gene inserts in the pED of BamHI, HindIII cutting through BamHI, HindIII cutting, forms the recombiant plasmid pED-β HCGCTP37 of amalgamation and expression, and the recombinant plasmid transformed e. coli bl21 obtains the recombination engineering bacteria.
5.Hh the structure of recombination engineering bacteria
The HSP65 gene inserts in the pED-β HCGCTP37 of NcoI, BamHI cutting through NcoI, BglII cutting, and the recombiant plasmid HSP65-β HCGCTP37 that forms amalgamation and expression is called for short Hh, and the recombinant plasmid transformed e. coli bl21 obtains the recombination engineering bacteria.
6.HX
2nThe structure of h recombination engineering bacteria
X
2Gene inserts in the Hh of NheI cutting through XbaI, NheI cutting, forms the recombiant plasmid HX of amalgamation and expression
2H still leaves single site NheI, and repetitive operation can be inserted one section X more on this basis
2Gene promptly inserts X
2Gene then obtains the recombiant plasmid HX of amalgamation and expression for n time
2nH, the recombinant plasmid transformed e. coli bl21 obtains the recombination engineering bacteria.
7. engineering bacterium fermentation and reorganization HSP65-β HCGCTP37 and HSP65-X
6The proteic acquisition of-β HCGCTP37
With LB is basal medium, and the Semen Maydis pulp culture medium is a fermentation medium, and fermentation parameter is as follows: temperature 36-38 ℃, and pH6.8-7.2.The centrifugal collection engineering bacteria in fermentation back, multigelation cracking thalline, centrifugal acquisition supernatant soluble protein, ammonium sulfate precipitation, contain the purpose fusion rotein in the 20%-40% ammonium sulfate precipitation, precipitation dissolving again can be judged the purity of the HSP65 of purification after the ion-exchange chromatography purification is used sds polyacrylamide gel electrophoresis.
In a fourth aspect of the present invention is the purposes of the anti-HCG polypeptide vaccine of adjuvant-free, and as mentioned above, the anti-HCG polypeptide vaccine of adjuvant-free can be used for the treatment of breast carcinoma, colon cancer, cancer of pancreas and carcinoma of prostate, also can be used for contraception and animal's castration.
Description of drawings
Fig. 1, vector plasmid pET28a-HSP65, and recombiant plasmid Hh and HX
6H.
Fig. 2. gene recombinant antigens HSP65-X
6The base sequence of-β HCGCTP37 (1953bp).
Fig. 3 .HX
6The β HCGCTP37 and the X of h plasmid
6And HSP C end parts gene sequencing result.
Fig. 4 .SDS-PAGE electrophoresis showed HSP65-β HCGCTP37 and HSP65-X
6The amalgamation and expression of-β HCGCTP37.1. standard molecular weight albumen; 2. e. coli bl21 cell whole protein; 3. the e. coli bl21 cell whole protein of load pED plasmid; 4. the e. coli bl21 cell whole protein of load pED-β HCGCTP37 plasmid; 5. the e. coli bl21 cell whole protein of load pET28a plasmid; 6. the e. coli bl21 cell whole protein of load pET28a-HSP65 plasmid; 7. the e. coli bl21 cell whole protein of load Hh plasmid; 8. load HX
2The e. coli bl21 cell whole protein of h plasmid; 9. load HX
4The e. coli bl21 cell whole protein of h plasmid; 10. load HX
6The e. coli bl21 cell whole protein of h plasmid.
Fig. 5 .SDS-PAGE electrophoresis showed HSP65-β HCGCTP37 and HSP65-X
6The proteic purification .1. of-β HCGCTP37 standard molecular weight albumen; 2. the e. coli bl21 cell whole protein of load pET28a plasmid; 3. load HX
6The e. coli bl21 cell whole protein of h plasmid; 4. the e. coli bl21 cell whole protein of load Hh plasmid; 5. isolating HSP65-X
6-β HCGCTP37; 6. isolating HSP65-β HCGCTP37.The A arrow shows HSP65-X
6The position of-β HCGCTP37; The B arrow shows the position of HSP65-β HCGCTP37.
Fig. 6 .ELISA measurement result shows to have only HSP65-X
6-β HCGCTP37 albumen can bring out mice and produce anti-HCG antibody.Legend: zero normal saline ◆ HSP ▲ Hh ■ HX
6h
Fig. 7 .EMT6 tumor growth curve shows through HSP65-X
6-β HCGCTP37 mice immunized is little through the normal mouse lotus tumor of HSP65-β HCGCTP37 mice immunized and injecting normal saline.Legend: zero normal saline ▲ Hh ■ HX
6h
Fig. 8 .EMT6 tumor mean size (figure A) and exemplary embodiment lock (figure B, legend: normal saline
Hh ■ HX
6H) show through HSP65-X
6-β HCGCTP37 mice immunized is than the normal mouse lotus tumor EMT6 little (P＜0.005) through HSP65-β HCGCTP37 mice immunized and injecting normal saline.
Fig. 9 .RM-1 inoculation mice time-survivor curve shows through HSP65-X
6-β HCGCTP37 mice immunized is the time-to-live long (P＜0.001) behind the normal mouse lotus tumor RM-1 of HSP65-β HCGCTP37 mice immunized and injecting normal saline.
Embodiment
Material:
(1) bacterial strain and plasmid:
This strain of escherichia coli (Escherichia coli AS1.357) obtains from Chinese DSMZ.Host bacterium E.coli BL21 is a genetic engineering common tool strain, at the laboratory relevant with genetic engineering research preservation is arranged all generally.
Plasmid pET28a is available from Novagen company.
Bacillus calmette-guerin vaccine is produced by Shanghai biological product company, contains Mycobacterium bovis (Mycobacterium bovis) in these goods.
Plasmid pED is made up and is preserved by this laboratory.
Plasmid pET28a-HSP65 is made up and is preserved by this laboratory, and HSP65 albumen is also by the separation and purification of this laboratory.
(2) enzyme and reagent:
Molecular cloning toolenzyme and reagent, bacterial genomes, plasmid extraction test kit are Pu Luomaige (Promega) company product.
(3) culture medium:
The LB culture medium, the document Sambrook J that sees reference that fills a prescription, Fristsh E F, Maniatis T.Molecular Cloning; A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989.This book is the classical works of technique for gene engineering, in many libraries of the universities collection is arranged all.
The Semen Maydis pulp culture medium contains: Semen Maydis pulp 25g/L, beef extract 15g/L, monosodium glutamate 10g/L.
Method
The molecular biology operational approach
The recovery of plasmid extraction, polymerase chain reaction, endonuclease digestion, dna segment, connection and transformed into escherichia coli: in the genetic engineering research field, these all are the routine operation methods, referring to Sambrook J, Fristsh E F, Maniatis T.Molecular Cloning; A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989, pp.16-340.
The mensuration of expression of recombinant proteins amount: referring to Sambrook J, Fristsh E F, Maniatis T.Molecular Cloning; A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989, method is carried out.
According to the aminoacid sequence of HSP65 gene and β HCGCTP37 polypeptide gene, select the codon of escherichia coli preferences for use, under computer auxiliary, design 8 oligonucleotide fragments.At first by the synthetic β HCGCTP37 polypeptide gene of PCR method, with the C end of this gene clone to the L-ansB-C gene of pED, transformed into escherichia coli obtains recombiant plasmid called after pED-β HCGCTP37.Be template with the pET28a-HSP65 plasmid again, by the method acquisition HSP65 gene of PCR, with the L-ansB-C gene among this gene replacement pED-β HCGCTP37, transformed into escherichia coli obtains recombiant plasmid called after Hsp65-β HCGCTP37 (being called for short Hh).Obtain gene (the abbreviation X that β HCG109-118 polypeptide repeats arranged in series for twice by the PCR method then
2), between the Hsp65 gene and β HCGCTP37 gene of Hh, transformed into escherichia coli obtains recombiant plasmid called after H X with this gene clone
2H; Repeated cloning X
2Gene obtains recombiant plasmid H X
4H; Repeated cloning X again
2Gene obtains recombiant plasmid H X
6H.Concrete grammar is as follows:
P1：5’GAC?GAC?CCG?CGT?TTC?CAG?GAC?TCT?TCT?TCT?TCT?AAA?GCT?CCG?CCG?CCA?TCT?CTG?3’
P2：5’AGT?GTC?AGA?CGG?ACC?CGG?CAG?ACG?AGA?CGG?AGA?CGG?CAG?AGA?TGG?CGG?CGG?AGC3’
P3：5’GGGG?GAT?CCG?ACT?GGT?GGC?ACT?TGC?GAC?GAC?CCG?CGT?TTC?CAG?GAC3’
P4：5’AAAA?AGC?TTA?CTG?CGG?CAG?GAT?CGG?AGT?GTC?AGA?CGG?ACC?CGG?CAG3’
P5：5’TTG?ACC?ATG?GCC?AAG?ACA?ATT?GCG?TAC3’
P6：5’TAA?AAG?ATC?TGC?GCT?AGC?GAA?ATC?CAT?GCC?ACC?CAT?3’
P7：5’ATA?TCT?AGA?ACT?TGC?GAC?GAC?CCG?CGT?TTC?CAG?GAC?TCT?ACT?TGC?GAC?GAC?CCG3’
P8：5’AAA?GCT?AGC?AGA?GTC?CTG?GAA?ACG?CGG?GTC?GTC?GCA?AGT?TGA?GTC?CTG?GAA?ACG3’
Obtain β HCG CTP37 polypeptide gene by PCR: first step oligonucleotide fragment P1 and P2 mix by a certain percentage, and the two is primer and template each other, and 94 ℃, 1min, 58 ℃, 45sec, 72 ℃, 45sec 15 takes turns totally; 72 ℃, 10min.Be template with this PCR product again, carry out pcr amplification with P3 and P4 primer, 94 ℃, 1min, 58 ℃, 1min, 72 ℃, 1min 20 takes turns totally; 72 ℃, 10min.The PCR product that amplification is obtained digests with restricted enzyme BamH1 and HindIII, be connected with pED plasmid with the digestion of same restrictions restriction endonuclease, connect product transformed into escherichia coli Escherichia coli BL21 (DE3), contain the recombiant plasmid pED-β HCGCTP37 that is connected with β HCG CTP37 polypeptide amalgamation protein gene in the transformant that is obtained.
Obtain HSP65-β HCGCTP37 polypeptide gene by PCR: with P5 is forward primer, P6 is a downstream primer, the pET28a-HSP65 plasmid is a template, through 94 ℃ of degeneration 5min, after carrying out 35 circulations altogether with 94 ℃ of degeneration 1min, 62 ℃ of renaturation 2min, 72 ℃ of extension 4min then, 72 ℃ are extended 10min again.The PCR product that amplification is obtained digests with restricted enzyme NcoI and BglII, be connected with pED-β HCGCTP37 plasmid with the digestion of same restrictions restriction endonuclease, connect product transformed into escherichia coli Escherichia coli BL21 (DE3), contain the recombiant plasmid Hh of HSP65-β HCGCTP37 polypeptide amalgamation protein gene in the transformant that is obtained, its structural framing is seen Fig. 1.
Obtain HSP65-X by PCR
6-β HCGCTP37 polypeptide gene: mix by a certain percentage with oligonucleotide fragment P7 and P8, the two is primer and template each other, through 94 ℃ of degeneration 5min, then with 94 ℃ of degeneration 1min, 58 ℃ of renaturation 50sec, 72 ℃ extend 30sec and carry out 30 circulations altogether after, 72 ℃ are extended 10min again.PCR product X with the amplification acquisition
2With restricted enzyme XbaI and NheI digestion, be connected with the Hh plasmid that digests with restricted enzyme NheI, connect product transformed into escherichia coli BL21, contain HSP65-X in the transformant that is obtained
2The recombiant plasmid HX of-β HCGCTP37 polypeptide amalgamation protein gene
2H.Restricted enzyme XbaI and NheI are isocaudarners, and enzyme connects the bonding site of back formation no longer by XbaI or NheI identification, so recombiant plasmid H X
2Still only leave a NheI restriction enzyme site among the h.Again with pcr amplification product X
2With restricted enzyme XbaI and NheI digestion, with H X with restricted enzyme NheI digestion
2The h plasmid connects, and connects product transformed into escherichia coli BL21, contains HSP65-X in the transformant that is obtained
4The recombiant plasmid H X of-β HCGCTP37 polypeptide amalgamation protein gene
4H; Carry out this process once more and obtain containing HSP65-X
6The recombiant plasmid H X of-β HCGCTP37 polypeptide amalgamation protein gene
6H, its structural framing is seen Fig. 1.Carrying out the sequence analysis of nucleic acid, further verify the correctness of GRF gene and reading frame thereof. sequencer map is seen Fig. 3.From sequencing result as can be seen, HSP65 sequence, six sections β HCG109-118 repetitive sequences and β HCGCTP37 sequence are connected, and have realized gene fusion.
With recombiant plasmid Hh and H X
6H is transformed into escherichia coli BL21 respectively.There is on the different transformant flat boards picking list bacterium colony kind go into the LB fluid medium from growth, spend the night 37 ℃ of shaken cultivation, go in the fresh Semen Maydis pulp fluid medium in 2% ratio transferred species, cultivated 4 hours for 37 ℃, the adding final concentration is that the IPTG of 0.1mmol/L induces escherichia coli expression T
7RNA polymerase, thus continue to cultivate expressed fusion protein.Induce the back bacterium liquid that took a morsel every 1 hour, centrifugal recovery thalline, SDS-PAGE electrophoresis show through thin slice scan has more realized HSP65-β HCGCTP37 polypeptide gene and HSP65-X
6The amalgamation and expression of-β HCGCTP37 polypeptide gene, and HSP65-X
2nThe proteic molecular weight ratio HSP65-X of-β HCGCTP37
2 (n-1)It is big by about 2400 that-β HCGCTP37 albumen is wanted.Fusion rotein reached stable maximum expression in back 6 hours inducing, and fusion rotein accounts for about 40% of total bacterial protein, the results are shown in Figure 4.
Engineering bacteria behind the abduction delivering is through centrifugal recovery thalline, thalline is suspended in shell-broken liquid (pH8.0 phosphate-buffered salt, 0.02% lysozyme) in, stirred 90 minutes at 30 ℃ after the freeze thawing, adding DNase digests DNA to solution not till the thickness, centrifugal recovery supernatant is used ammonium sulfate precipitation, and destination protein is mainly in the 20%-40% ammonium sulfate precipitation.Sedimentary fusion rotein is dissolved in the pH8.0 phosphate-buffered salt again, DEAE cellulose DE52 post (2 * 60cm) on the supernatant of centrifugal back, use the PBS/NaCl gradient elution, fraction collection carries out the SDS-PAGE electrophoresis detection, HSP65 is in the eluting peak at 120mmol place, prepare the purity of sample with SDS-PAGE electrophoretic examinations, see Fig. 5.
Select 7-8 BALB/C mice in age in week for use, HSP65-β HCGCTP37 experimental group, HSP65-X
6Each 8 of-β HCGCTP37 experimental group, HSP65 blank group and normal saline negative control group.All mices all carry out the pre-immunity of bacillus calmette-guerin vaccine (BCG) (Shanghai biological product company) except that the normal saline group, and freeze dried bacillus calmette-guerin vaccine is mixed with 5 * 10 with normal saline
6CFU/ml, every Corium Mus is injected 100 μ l down.After pre-immune two weeks, carry out the immunity first time, respectively HSP65-β HCGCTP37, the HSP65-X of subcutaneous injection 100 μ l (0.5 μ g/ μ l is in normal saline)
6-β HCGCTP37 and HSP65 albumen need not freund adjuvant emulsifyings.Later on once, carry out booster immunization altogether 2 times every 2 all booster immunizations.The normal saline of normal saline group injection same amount.Each immunity back 2 all endocanthions are got blood once, and blood volume is 0.5-0.8ml, and is centrifugal, gets the freezing preservation of serum; Pre-immunity was put to death rat after 80 days, and eyeball is got blood, the freezing preservation of centrifuging and taking serum.
Spend the night bag by 96 hole ELISA ELISA Plate with 4 ℃ of the pED-β HCGCTP37 fusion rotein of purification, and every hole 100 μ l contain the 100ng fusion rotein.Behind the bag quilt, sealed one hour in 37 ℃ of full holes with 5% bovine serum albumin (BSA) solution.Get blood gained antiserum after the immunity, with 100 times of 5%BSA (in the PBS of pH7.5) dilutions, the antiserum that every then hole adds after the 100 μ l dilution acts on 1 hour in 37 ℃.Add the goat anti-mouse igg two anti-(horseradish peroxidase-labeled) of 100 μ l with after PBST (the containing 0.1%Tween-20) washing 6 times, every hole 100 μ l, 37 ℃ of effects 1 hour with dilution in 1: 20000.PBST washing 6 times, every hole add 100 μ l hydrogen peroxide ureas and 3,3`, 5,5`-tetramethyl benzidine (TMB) solution and after 20 minutes, add 50 μ l 2MH in 37 ℃ of colour developings
2SO
4Cessation reaction, and under the 450nm wavelength, measure each hole absorbance.The results are shown in Figure 6.As can be seen from the results, the HSP65-X that only contains the repetition epitope
6-β HCGCTP37 albumen can bring out special anti-HCG antibody.
Select female BALB/C mice in 7-8 age in week for use, HSP65-β HCGCTP37 experimental group, HSP65-X
6Each 6 of-β HCGCTP37 experimental group and normal saline negative control group.All mices all carry out the pre-immunity of bacillus calmette-guerin vaccine (BCG) (Shanghai biological product company) except that the normal saline group, and freeze dried bacillus calmette-guerin vaccine is mixed with 5 * 10 with normal saline
6CFU/ml, every Corium Mus is injected 100 μ l down.After pre-immune two weeks, carry out the immunity first time, respectively HSP65-β HCGCTP37 and the HSP65-X of subcutaneous injection 100 μ l (0.5 μ g/ μ l is in normal saline)
6-β HCGCTP37 albumen need not freund adjuvant emulsifying.Later on once, carry out booster immunization altogether 2 times every 2 all booster immunizations.The normal saline of normal saline group injection same amount.Last immunity the 3rd week of back, experimental group and control group mice right side front subcutaneous vaccination 0.1mL (10
7Individual/mL) EMT6 mouse mastopathy cell.Observed and recorded tumor growth situation is with the long and short footpath of vernier caliper measurement tumor.Gross tumor volume is V (mm by formula
3)=0.4ab
2Calculate, a=long (mm), b=wide (mm), the comparison of tumor size is also drawn growth curve, sees Fig. 7.Behind tumor cell inoculation the 28th day, all animals eyeball was put to death after getting blood, gets liver,spleen,kidney and ovary uterus and tumor tissue, weighs, and takes pictures, and sees Fig. 8.Show through HSP65-X
6-β HCGCTP37 mice immunized is than the normal mouse lotus tumor EMT6 littler (P＜0.005) through HSP65-β HCGCTP37 mice immunized and injecting normal saline.
Select male C57/BL6 mice in 7-8 age in week for use, HSP65-β HCGCTP37 experimental group, HSP65-X
6Each 7 of-β HCGCTP37 experimental group and normal saline negative control group.All mices all carry out the pre-immunity of bacillus calmette-guerin vaccine (BCG) (Shanghai biological product company) except that the normal saline group, and freeze dried bacillus calmette-guerin vaccine is mixed with 5 * 10 with normal saline
6CFU/ml, every Corium Mus is injected 100 μ l down.After pre-immune two weeks, carry out the immunity first time, respectively HSP65-β HCGCTP37 and the HSP65-X of subcutaneous injection 100 μ l (0.5 μ g/ μ l is in normal saline)
6-β HCGCTP37 albumen need not freund adjuvant emulsifying.Later on once, carry out booster immunization altogether 2 times every 2 all booster immunizations.The normal saline of normal saline group injection same amount.Last immunity the 4th week of back, experimental group and control group mice prostate inoculation 0.02mL (2*10
7Individual/mL) RM-1 mice prostate gland cancer cell.Observed and recorded mice survival condition, and draw time-survivor curve, see Fig. 9.Show through HSP65-X
6-β HCGCTP37 mice immunized is the time-to-live longer (P＜0.001) behind the normal mouse lotus tumor RM-1 of HSP65-β HCGCTP37 mice immunized and injecting normal saline.
Claims (8)
1. method of making the strong immunogenic polypeptide vaccine of adjuvant-free is characterized in that downstream that two sections multiple antigen epitope polypeptide tandem genes are inserted the HSP65 gene repeatedly making the multiple multiple series connection of antigen epitope polypeptide.And the antigen epitope polypeptide gene is connected with the heatshock protein HSP65 gene that comes from mycobacteria (Mycobacterium bovis), makes antigen epitope polypeptide and HSP65 amalgamation and expression.
2. adjuvant-free anti-hCG polypeptide vaccine, this vaccine is made in accordance with the method for claim 1, it is characterized in that need not adding adjuvant, do not need antigen polypeptide and carrier chemical coupling yet, can excitating organism generation strong immunization reply, produce the anti-hCG antibodies of high titre, and, make the growth of body tumor cell be subjected to remarkable inhibition by the blocking-up human chorionic gonadotropin.
3. according to the described vaccine of claim 2, it is characterized in that containing in the vaccine polypeptide six sections human chorion gonadotrophic hormone beta chain 109-118 and repeat the tandem polypeptide section, its aminoacid sequence is ThrCysAspAspProArgPheGlnAspSerThrCysAspAspProArgPheGlnAs pSerAlaArgThrCysAspAspProArgPheGlnAspSerThrCysAspAspProA rgPheGlnAspSerAlaArgThrCysAspAspProArgPheGlnAspSerThrCys AspAspProArgPheGlnAspSerAlaSer, end two aminoacid AlaSer corresponding DNA sequences is the recognition site of DNA restricted enzyme Nhe I, can be used for inserting the antigen epitope polypeptide gene repeatedly, make the more former epitope peptide section of multi-resistance of connecting.The structure of vaccine polypeptide can be expressed as: HSP65-X
2n-β HCGCTP37 peptide inserts gene X
2N time, then contain 2n placed in-line human chorion gonadotrophic hormone beta chain 109-118 epitope peptide section in the vaccine polypeptide, n 〉=1.
4. according to the described vaccine of claim 2, the heatshock protein HSP65 amalgamation and expression that it is characterized in that polypeptide and Mycobacterium bovis (Mycobacterium bovis), fusion rotein produces intensive immunne response as vaccine excitating organism under the situation of additionally not adding adjuvant.
5. adjuvant-free anti-hCG polypeptide vaccine preparation method, comprising with chemical synthesis and the synthetic human chorion gonadotrophic hormone beta chain C of PCR method holds 37 peptides, two sections human chorion gonadotrophic hormone beta chain 109-118 to repeat the coded sequence DNA of tandem polypeptide section and HSP65, these DNA are inserted expression vector step by step, go out soluble fusion protein at expression in escherichia coli; Through the ammonium sulfate precipitation purification; Through ion exchange resin DEAE cellulose purification, obtain anti-hCG vaccine polypeptide again.
6. according to the described adjuvant-free anti-hCG of claim 4 polypeptide vaccine, it is characterized in that immune animal can bring out body and produce anti-hCG antibodies, combine with human chorionic gonadotropin by anti-hCG antibodies in the body, breast carcinoma, colon cancer, cancer of pancreas and carcinoma of prostate that human chorionic gonadotropin relies on be can treat, contraception and animal's castration also can be used for.
7. according to the described adjuvant-free anti-hCG of claim 4 polypeptide vaccine, it is characterized in that and to make pharmaceutical composition with the pharmaceutical carrier combination.
8. according to the described adjuvant-free anti-hCG of claim 4 polypeptide vaccine, it is characterized in that and to make pharmaceutical composition with the combination of other drug active component.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108611310A (en) * | 2018-04-28 | 2018-10-02 | 中国药科大学 | A kind of recombination Hsp65 and STEAP1186-193The structure of the genetic engineering bacterium of fusion protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108611310A (en) * | 2018-04-28 | 2018-10-02 | 中国药科大学 | A kind of recombination Hsp65 and STEAP1186-193The structure of the genetic engineering bacterium of fusion protein |
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