CN101905018A - Recombinant fusion protein vaccine and attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection - Google Patents

Recombinant fusion protein vaccine and attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection Download PDF

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CN101905018A
CN101905018A CN2010101396421A CN201010139642A CN101905018A CN 101905018 A CN101905018 A CN 101905018A CN 2010101396421 A CN2010101396421 A CN 2010101396421A CN 201010139642 A CN201010139642 A CN 201010139642A CN 101905018 A CN101905018 A CN 101905018A
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vaccine
fusion protein
vaca
ureb
caga
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CN101905018B (en
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邹全明
刘开云
吴超
毛旭虎
郭刚
石云
余抒
陈立
解庆华
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Third Military Medical University TMMU
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Abstract

The invention relates to a recombinant fusion protein vaccine and an attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection, and belongs to the field of biopharmaceutics. The recombinant fusion protein vaccine and the attenuated live vector vaccine for expressing the recombinant fusion protein are characterized in that: the recombinant fusion protein is formed by connecting immune protective function fragments of helicobacter pylori cytotoxin relevant gene protein CagA from helicobacter pylori, vacuolization cytotoxin VacA and urease subunit UreB linearly, and the immunogenicity and immune protection of the recombinant fusion protein are verified through animal experiments. The live vaccine has the advantages that: 1, an Hp fusion protein gene can be expressed stably; 2, mucosa and systemic immune response can be induced after immunity; and 3, the method is convenient, the cost is low, and the economic benefit is obvious. Therefore, the attenuated live vector vaccine can be used as candidate vaccines for treating and preventing the Hp infection.

Description

The recombination fusion protein vaccine and the attenuated live vector vaccine of treatment and prevention helicobacter pylori infections
Technical field
The present invention relates to field of biological pharmacy, the recombination fusion protein vaccine and the attenuated live vector vaccine of particularly a kind of treatment and prevention helicobacter pylori infections.
Background technology
Gastritis and peptic ulcer are human commonly encountered diseases and frequently-occurring diseases, and gastric cancer also is one of higher malignant tumor of M ﹠ M.Helicobacter pylori (Helicobacter pylori, Hp) be the main cause of disease of chronic active gastritis, peptic ulcer, simultaneously relevant closely related (the Nomura A of the lymphadenomatous generation of lymphoid tissue with adenocarcinoma of stomach and gastric mucosa, Stemmermann GN, Chyou PH et al.Helicobacter pylori infection and gastric carcinoma among JapaneseAmericans in Hawaii.N-Engl-J-Med.1991,325 (16): 1132-1136.; Parsonnet J, Friedman GD, Vandersteen DP, et al.Helicobacter pylori infection and the risk of gastric carcinoma.N-Engl-J-Med.1991,325 (16): 1127-1131.; Dunn BE, Cohen H, Blaser MJ.Helicobater pylori.Clin-Microbiol-Rev.1997,10 (4): 720-741.; Parsonnet J, Shmuely H, Haggerty T.Fecal and oral shedding of Helicobacter pylori from healthy infected adults.J-Am-Med-Assoc.1999,282 (23): 2240-2245.), World Health Organization (WHO) has classified Hp as I class cancerigenic factor.A large amount of epidemiological study data show that Hp infects and is distribution on global.In developed country, the Hp infection rate is about 40～50%, and can reach 50～60% (Lee A.Prevention of Helicobacter pylori infection.Scand-J-Gastroenterol.1996.Suppl 215:11-15.) in developing country.According to statistics, be 58.07% at the average infection rate of China (wangkai is beautiful, Wang Runtian. and Chinese helicobacter pylori infections epidemiology Meta analyzes. Chinese epidemiology magazine .2003,24 (6): 443-446.).Proton pump inhibitor adds the Hp eradication therapy one line scheme that two kinds of antibiotic triple therapies are whole world recommendations at present, but along with the extensive use of antibiotic in the Hp treatment of infection, the incidence rate of Hp persister constantly rises, so that the difficulty that Hp eradicates increasing (Hu Fulian. helicobacter pylori drug resistance and treatment Failure Causes Analysis. Chinese digestive endoscopy .2009,3 (3): 1-8.).And have that expense costliness, eradication rate are low, the recurrence after the drug withdrawal with infect again, patient's problems such as compliance difference.
Vaccine is the most cost-effective way of sense of control metachromia disease, produces by the boosting vaccine body to be different from the specific immune response that natural infection causes, and can reach the purpose that prevention or treatment Hp infect.Competitively study the Hp vaccine both at home and abroad, researchs such as the existing so far full bacterium inactivated vaccine of Hp, genetic engineering subunit vaccine, attenuated live vector vaccine, nucleic acid vaccine, at present, " oral reorganization Hp vaccine " (traditional Chinese medicines card word: S20090002) succeed that only has us to develop.
Urease is a kind of metalloenzyme of energy hydrolyze urea, accounts for the 8%-10% of Hp tropina total amount, and is relevant in gastric field planting ability with Hp, and the cell effect that can cause inflammation, the damage gastric epithelial cell.Urease B subunit (UreB) is the first-selected protective antigen (Lee A.Vaccination against Helicobacter pylori.J-Gastroenterol.1996,31 Suppl 9:69-74.) of present vaccine Hp research.Cytotoxin-associated gene albumen (CagA) is one of important virulence factor of Hp, and the CagA positive strain infects the danger that takes place than serious gastrointestinal disease such as easier increase duodenal ulcer of negative strain and gastric cancer.Vacuolating cytotoxin A (VacA) is a kind of Hp toxin, it can cause that gastric epithelial cell generation cavity sample becomes, cell death inducing, suppressor T cell propagation, be one of the factor that infects of the long-term field planting of Hp (Backert S, Meyer TF.Type IV secretion systems and their effectors in bacterial pathogenesis.Curr-Opin-Microbiol.2006,9 (2): 207-217.; Amieva MR, El-Omar EM.Host-bacterialinteractions in Helicobacter pylori infection.Gastroenterology.2008,134 (1): 306-323.).HpCagA, VacA and UreB all have good antigenicity, can reduce the field planting level of Hp in gastric tissue by the mode of mucosal immunity significantly, show the certain protection effect.In the bacterial infection process, because the complexing action mechanism between host and the pathogenic bacterium, the vaccine of single antigen component is difficult to produce fully and effectively protective effect.Existing studies show that, the antigenic immune protective rate of single Hp almost all is lower than 80%, the antigen combination can obtain ideal protection effect (Telford JL more than 2 kinds and 2 kinds, Ghiara P.Prospects for the development of a vaccine against Helicobacterpylori.Drugs.1996,2 (6): 799-804.; Ruggiero P, Peppoloni S, Berti D, et al.New strategies forthe prevention and treatment of Helicobacter pylori infection.Expert-Opin-Investig-Drugs.2002,11 (8): 1127-1138.).The protective antigen relative molecular mass that is used for the Hp vaccine is bigger, and it is beyond expression of words or expression rate is extremely low to make up complete genetic engineering recombination fusion protein.The protein fragments of selecting to have immunity protection function in the high molecular weight protein substitutes the full-length proteins molecule, still can bring into play effective immunne response effect, and offers convenience for the structure of genetic engineering recombiant vaccine.
The field planting environment that Hp is special has determined that mucosal immunity is a vaccine immunity mode preferably, and mucosal immunity not only can excite intensive humoral immunization, can also induce the rising of partial IgA antibody horizontal simultaneously preferably.Mucomembranous immune system is the important component part of body immune system, mainly comprises intestinal mucosa associated lymphoid tissue and bronchial mucosa associated lymphoid tissue etc., and it is bringing into play unique effect aspect the body defense function.By mesenteric lymph node, and the big amount lymphocyte that is dispersed in proper mucous membrane and the enteric epithelium is formed immune induction position and immunological effect position.The various immunocytes of mucomembranous surfaces such as gastrointestinal tract are by absorbing, process and offer antigen, produce justacrine at this antigenic antibody (being mainly slgA), the latter can with carry this antigenic carrier such as antibacterial, virus etc. the specificity association reactions take place, stop its field planting mucomembranous surface or invasion and attack body, thereby play certain immunologic defence function.But, the greatest drawback of mucosal immunity is easily to resist former generation immunologic tolerance, even improve the antigen amount, the antigenic specificity slgA level that body or mucosa produce is but very low, to the infection by microorganisms of carrying corresponding antigens almost do not have the immune defence ability or extremely weak, very easily cause the generation of immunologic tolerance.
Salmonella typhimurtum is a bacterium in a kind of common aggressive born of the same parents, by behind the gene engineering method attenuation to the pathogenic remarkable reduction of host, but still keep good aggressivity, can be after oral at the intestinal parasitized breeding, can be by small intestinal M cellular uptake, pass the enteric epithelium barrier, also given immune response (the Sirard JC of immunocyte inducing specific simultaneously by APC cell submission because of its bacterial antigens, Niedergang F, Kraehenbuhl JP.et al.Live attenuated Salmonella:aparadigm of mucosal vaccines.Immunol-Rev.1999,171:5-26.), the ideal carrier that is considered to mucosal immunity is one of at present the most frequently used attenuated live vaccine carrier.That the attenuated live carrier bacterin has is safe, can be taken orally, guard time is long, side effect is little, cheap, be easy to advantages such as production, storage and transportation.At present existing a plurality of recombinant attenuated Salmonella typhimurtum vaccines enter clinical research (Konadu EY, Lin FY, et al.Phase 1 and phase 2 studies ofSalmonella enterica serovar paratyphi A O-specific polysaccharide-tetanus toxoid conjugates inadults, teenagers, and 2-to 4-year-old children in Vietnam.Infect-Immun.2000,68 (3): 1529-1534.; Tacket CO, Sztein MB, et al.Phase 2 clinical trial of attenuated Salmonella entericaserovar typhi oral live vector vaccine CVD 908-htrA in U.S.volunteers.Infect-Immun.2000,68 (3): 1196-1201.; Cunningham C, Nemunaitis J.A phase I trial of genetically modifiedSalmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administeredby intratumoral injection in combination with 5-fluorocytosine for patients with advanced ormetastatic cancer.Protocol no:CL-017.Version:April 9,2001.Hum-Gene-Ther.2001,12 (12): 1594-1596.; Toso JF, Gill VJ, et al.Phase I study of the intravenous administration of attenuatedSalmonella typhimurium to patients with metastatic melanoma.J-Clin-Oncol.2002,20 (1): 142-152.).Can not there be the drug resistance plasmid in regulation according to U.S. food and medication management mechanism (FDA) in the live vaccine, and also can't usually keep the stability of recombiant plasmid in the humans and animals body with antibiosis.In order not have under the antibiotic existence condition, the external source protective antigen can be in the attenuation Salmonella typhi stably express, a kind of novel plasmid vector system, i.e. carrier-host's balanced lethal system have grown up at present.Balanced lethal system is a kind of deadly system of plasmid-balance of chromosome that is masked as principal character with nutrition selection marker replacement antibiotic resistance, also be carrier-host's balanced lethal system (Tacket CO, Kelly SM, Schodel F.et al.Safety and immunogenicity in humans of an attenuated Salmonellatyphi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by theAsd-balanced lethal vector system.Infect-Immun.1997,65 (8): 3381-3385.; Chen H, M.SchifferliD.Mucosal and systemic immune responses to chimeric fimbriae expressed by Salmonellaenterica serovar typhimurium vaccine strains.Infect-Immun.2000,68 (6): 3129-3139.).Aspartic acid β semialdehyde dehydrogenase gene (asd) disappearance among the attenuated salmonella typhimurium strain strain χ 4550, because the aspartic acid β semialdehyde dehydrogenase of this asd gene code is the indispensable enzyme in meso diaminopimelic acid (DAP) biosynthesis pathway, and DAP is the ultimate constituent of gram-negative bacteria whole cell peptidoglycan, therefore the sudden change of asd gene causes the synthetic shortage of DAP, causes this mutant to become auxotrophic strain.Under the condition of culture of no exogenous DAP, bacteriolyze death.This bacterial strain is as being contained asd expression of gene plasmid by being changed over to, and because of carrier and deficiency host bacterium formation genetic complementation, then this bacterial strain can be stablized and carries the plasmid expression of going down to posterity.Under this condition, it is essential that recombiant plasmid becomes bacteria live institute, in case the transformant plasmid loss, antibacterial then can't normally grow on the culture medium owing to can not synthesize essential material.Therefore, every antibacterial that can grow on normal culture medium must comprise recombiant plasmid, thereby constitute balanced lethal system.The attenuation salmonella live vaccine that utilizes " carrier-host's balanced lethal system " to make up demonstrates good prospects for application especially.
In sum, fully fully utilize the antigenic characteristic of Hp CagA, VacA and UreB, these several antigenic characteristics are worked in coordination with to be brought into play, and overcome the problems such as easy generation toleration that it exists aspect mucosal immunity, a kind of vaccine that is used for the treatment of and prevents Hp to infect is provided, to bring greatest Gospel to gastritis and peptic ulcer patient, but not have the report of this respect at present.
Summary of the invention
The present invention is directed to the blank in above-mentioned field, the recombination fusion protein vaccine and the live vaccine of a kind of treatment and prevention helicobacter pylori infections is provided.
The recombination fusion protein vaccine of a kind of treatment and prevention helicobacter pylori infections; comprise one section recombination fusion protein, it is characterized in that described recombination fusion protein origin comes from helicobacter pylori cytotoxin related gene PROTEIN C agA, the vacuolating cytotoxin VacA of helicobacter pylori and the immunity protection function fragment linearity that has of urease subunit UreB connects and composes.
The immunity protection function fragment CagA of described helicobacter pylori cytotoxin related gene PROTEIN C agA 160Aminoacid sequence shown in Seq ID No.1; The immunity protection function fragment VacA of described vacuolating cytotoxin VacA 62Aminoacid sequence shown in Seq ID No.2; The immunity protection function fragment UreB of described urease subunit UreB 138Aminoacid sequence shown in Seq ID No.3.
Described recombination fusion protein vaccine has CagA 160-VacA 62-UreB 138The order of connection.
The CagA of described recombination fusion protein vaccine 160, VacA 62, UreB 138Between connection adopt connection peptides.
Described recombination fusion protein vaccine has the aminoacid sequence shown in Seq ID No.4
The encode gene of above-mentioned recombination fusion protein vaccine.
Described gene has nucleotide sequence shown in the Seq ID No.5.
A kind of recombiant plasmid comprises skeleton carrier and exogenous gene, and exogenous genetic fragment comprises the gene of the above-mentioned recombination fusion protein vaccine of encoding.
The skeleton carrier of described recombiant plasmid is for containing the segmental Salmonella typhimurium bacteria plasmid of aspartic acid β semialdehyde dehydrogenase gene pYA3149.
The attenuated live vector vaccine of a kind of treatment and prevention helicobacter pylori infections, comprise carrier bacterium and express immunogenic exogenous plasmid, described carrier bacterium is an attenuated live, described exogenous plasmid is above-mentioned recombiant plasmid, described attenuated live refer to by behind the gene engineering method attenuation to host's pathogenic remarkable reduction, and kept bacterial strain to the infection ability of the mucosa of stomach, intestinal or cell.
Described attenuated live is the attenuated salmonella typhimurium strain strain.
Described attenuated salmonella typhimurium strain strain is the bacterial strain χ 4550 that has lacked aspartic acid β semialdehyde dehydrogenase gene.
The preparation method of the attenuated live vector vaccine of above-mentioned treatment and prevention helicobacter pylori infections, step is as follows:
(1) with the helicobacter pylori genome be template, amplification has the genetic fragment of nucleotide sequence shown in Seq ID No.12, Seq ID No.13, the Seq ID No.14 respectively.
(2) ligation amplification obtains three genetic fragments, and is building up to and contains on the segmental skeleton carrier pYA3149 of aspartic acid β semialdehyde dehydrogenase gene, the screening recombiant plasmid;
(3) recombiant plasmid changes host bacterium X over to 4550In,
The primer of the genetic fragment of nucleotide sequence is as follows respectively shown in amplification Seq ID No.12, Seq ID No.13, the Seq ID No.14:
cagA 160P1/cagA 160P2：
5-GCG
Figure GSA00000053969900051
GAAACGCTCAATCAAGAAC-3，
5-GCTACCTCCTCCACTTCCGCCTCCTTGTGAGCCTGTGAGTTGGTCTTC-3；
vacA 62P1/vacA 62P2：
5GGAGGCGGAAGTGGAGGAGGTAGCTTAGGCACTAACAGCATTAGTAATGT3，
5GCTACCTCCTCCACTTCCGCCTCCGATAAGATACTTGTAATTGTCGGGGT3；
ureB 138P1/ureB 138P2：
5GGAGGCGGAAGTGGAGGAGGTAGCGACACTTTGAATGAAGCTGGTTGTG3，
5GCGAAGCTTAAAATTCTTTCTTGTTTTTGTCAG3。
The object of the present invention is to provide the vaccine of treatment and prevention helicobacter pylori infections; from Hp 26695, screen CagA, VacA and UreB albumen has the fragment of immunity protection function; by molecular biology method clone recombination; make up recombinant expression plasmid, express obtaining recombination fusion protein.From the result of embodiment 4 as can be seen, this recombination fusion protein has good immunogenicity, can the stimulation test Mus produce the specific immune response of anti-HP, and the experimental mouse of oral empty plasmid transformed bacteria bacterium does not produce the antibody of anti-HP.Those skilled in the art are based on having now in the document CagA, VacA and three kinds of proteic reports of UreB; may select the different fragments that immunologic function is arranged, make up the expressed recombination fusion protein vaccine of the carrier that gets all in protection scope of the present invention according to technical scheme provided by the invention then.Among the present invention, the segmental optimal choice that CagA, VacA and UreB albumen is had immunity protection function is as follows: CagA 160Proteic aminoacid sequence such as GenBank:NP_207343.1 (231-390 amino acids, Seq ID No.1); VacA 62Proteic aminoacid sequence such as GenBank:NP_207680.1 (744-805 amino acids Seq ID No.2); UreB 138Proteic aminoacid sequence such as GenBank:NP_206872.1 (250-387 amino acids Seq ID No.3).
CagA wherein encodes 160, VacA 62And UreB 138Nucleotide sequence be respectively: cagA 160, vacA 62And ureB 138
CagA 160Cover the nucleotide sequence 688-1167 position (SeqID No.12) of the Hp 26695 bacterial strain HP0547 genes of GenBank announcement, comprised the upstream restriction enzyme site and the downstream Linker nucleotide sequence total length 513bp of introducing.
VacA 62Cover the nucleotide sequence 2230-2415 position (SeqID No.13) of the Hp 26695 bacterial strain HP0887 genes of GenBank announcement, comprised the upstream and downstream Linker nucleotide sequence total length 234bp of introducing.
UreB 138Cover the nucleotide sequence 748-1161 position (SeqID No.14) of the Hp 26695 bacterial strain HP0072 genes of GenBank announcement, comprised the upstream Linker and the downstream restriction enzyme site nucleotide sequence total length 448bp of introducing.
In the recombination fusion protein that the present invention makes up, CagA 160, VacA 62And UreB 13Connect with linear arrangement, and its immune effect the best during with the sequence arrangement of CagA160-VacA62-UreB138; The present invention is further preferably at CagA 160, VacA 62And UreB 13The place of interconnecting of three elements adds (GGGS) 2～3, (GGGGS) 2～3, (RSIAT) 2～3Or the RPACKIPNDLKQKVMNH peptide chain is flexible Linker, and those skilled in the art can require other multiple connection peptides of design according to the routine of Linker, can both arrive purpose of the present invention.Preferred amino acid sequence of the present invention is GGGSGGGS (GeorgeRA, Heringa J.An analysis of protein domain linkers:their classification and role inprotein folding.Protein Engineering, 2002,15 (11): peptide chain 871-879.) is a connection peptides.
The present invention also provides the gene order of the above-mentioned recombination fusion protein of encoding, and is wherein a kind of shown in Seq ID No.5.Those skilled in the art can construct recombiant plasmid, the engineering bacteria of express recombinant amalgamation protein vaccine according to the invention provides gene order, are used for the claimed recombination fusion protein vaccine of production the present invention.
The skeleton carrier of a kind of recombiant plasmid that the present invention makes up adopts and contains the segmental Salmonella typhimurium bacteria plasmid of aspartic acid β semialdehyde dehydrogenase gene pYA3149, this recombiant plasmid can be used for being transferred to a kind of engineering bacteria with carrier-host's balanced lethal system of structure in the attenuation bacterium that lacks aspartic acid β semialdehyde dehydrogenase gene, with suitable vaccine as the human or animal.
A significant contribution of the present invention provides the attenuated live vector vaccine of a kind of treatment and prevention helicobacter pylori infections, is to adopt attenuated live as carrier bacterium, and be the engineering bacteria that exogenous plasmid makes up with the plasmid of above-mentioned express recombinant fusion rotein.The attenuated live that adopts refers to knock out its Disease-causing gene by artificial as gene engineering method, and to make its pathogenic reduction greatly to the people be attenuation, and kept the antibacterial of the infecting potential of the intestines and stomach tissue to the human or animal, mucosa, cell.Such engineering bacteria is as vaccine; can carry and express that immunogenic exogenous gene is colonizated in human or animal's gastrointestinal tract and not pathogenic; immune response takes place and synthesizes at immunogenic specific antibody in stimulating gastrointestinal road tissue; the protection body is avoided having identical immunogenic bacteria attack and is caused a disease; because live vaccine is colonizated in stomach intestinal tissue for a long time; can stimulate body to keep secular antibody synthesis capability; therefore the disposable immunity than inactivated vaccine has the better protection effect, is particularly suitable for prevention and the microbial gastritis of treatment helicobacter pylorus; the disease of this easy repeated infection outbreak of enteritis.
The present invention preferably adopts the salmonella typhi of attenuation as carrier bacterium, obtains the attenuated live vector vaccine after changing the recombiant plasmid that the present invention makes up over to.Attenuated salmonella typhimurium strain is a bacterium in a kind of common aggressive born of the same parents; by behind the gene engineering method attenuation the pathogenic remarkable reduction of host still being kept good aggressivity; and energy stable expression of exogenous gene; can be directly used in the immunoprotection test; do not need the expressed target antigen of purification; removed the complicated procedures of forming of protein post processing from; oral back per os; nose; mucosal route immunity such as rectum can be induced powerful and special immunoreation; can be by small intestinal M cellular uptake; pass the enteric epithelium barrier; also given the immune response of immunocyte inducing specific simultaneously by APC cell submission because of its bacterial antigens; the ideal carrier that is considered to mucosal immunity is one of at present the most frequently used attenuated live vaccine carrier.
Because attenuated live vector vaccine provided by the invention is mainly used in prevention and treatment HP infects, consider that the live vaccine that is used for human body or animal can not contain resistant gene, therefore can't adopt antibiosis usually to screen the positive engineering bacteria of structure, so the present invention preferably adopt salmonella typhi bacterial strain (Salmonella typhimurium χ 4550/Asd -Cya -Nal rCrp -) as carrier bacterium, because its sudden change has lacked aspartic acid β semialdehyde dehydrogenase gene (asd), the meso diaminopimelic acid (DAP) of external source need be provided, otherwise because of can't normally synthesizing cell wall bacteriolyze death takes place, need change the exogenous plasmid that carries the asd gene over to and just can grow on normal culture medium, therefore be a kind of ideal auxotroph screening bacterial strain.Changed the recombiant plasmid that contains the asd gene among the present invention over to,, obtained the engineering bacteria of the dead system of a kind of tool carrier-host's balance, met the standard of oral live vaccine, can be widely used in the infection of treatment and prevention HP as being the recombiant plasmid of skeleton carrier with pYA3149.Experimental data shows: this vaccine has lot of advantages: 1. can stably express Hp antigen-4 fusion protein gene; 2. can induce mucosa and systemic immunity to reply after the immunity; 3. safe and effective, method is easy, and cost is low, has remarkable economic efficiency.With this bacterial strain immunity Hp mice infected, mice can generate the specific antibody (Fig. 8) of anti-Hp, and can remove mice gastric Hp or reduce mice gastric Hp field planting amount (table 1, table 2), so the present invention can be used as the candidate vaccine of treatment and prevention Hp infection.
The present invention also provides the preparation method of above-mentioned live vaccine, and it mainly may further comprise the steps:
(1) by PCR method, from Hp 26695 genomic DNAs, clones CagA respectively 160, VacA 62And UreB 138Dna fragmentation;
(2) method of the overlapping PCR of employing, the dna fragmentation that clone in the step (1) is obtained connects into fusion gene;
(3) fusion gene is building up on the vector plasmid, order-checking is identified;
(4) fusion gene is building up on the expression vector, transforms host strain χ 4550 (Salmonella typhimurium χ 4550/Asd -Cya -Nal rCrp -), obtain recombinant attenuated Salmonella typhimurtum live vaccine;
Description of drawings
Fig. 1 .cagA160, vacA62 and ureB138 nucleic acid agarose gel electrophoresis figure.
Wherein swimming lane 1 is nucleic acid (DNA) molecular weight standard (TaKaRa, 100bp DNA Ladder Marker, D505A), swimming lane 2 is cagA160PCR amplified production (513bp), swimming lane 3 is vacA62PCR amplified production (234bp), and swimming lane 4 is ureB138PCR amplified production (448bp).
Fig. 2. be the agarose gel electrophoresis figure of cagA160-vacA62
Wherein swimming lane 1 is that (D505A), swimming lane 2 is the pcr amplification product (723bp) of cagA160-vacA62 fusion gene to nucleic acid (DNA) molecular weight standard for TaKaRa, 100bp DNA Ladder Marker.
The agarose gel electrophoresis figure of Fig. 3 .cagA160-vacA62-ureB138,
Wherein swimming lane 1 is that (D505A), swimming lane 2 is the pcr amplification product (1147bp) of cagA160-vacA62-ureB138 fusion gene to nucleic acid (DNA) molecular weight standard for TaKaRa, 100bp DNA Ladder Marker.
Fig. 4. be recombiant plasmid pET 22b (+)-cagA 160-vacA 62-ureB 138Enzyme action identify agarose gel electrophoresis figure
Wherein swimming lane 1 is that (D518A), swimming lane 2 is recombiant plasmid pET 22b (+)-cagA to nucleic acid (DNA) molecular weight standard for TaKaRa, Wide Range DNA Marker (100～6,000) 160-vacA 62-ureB 138EcoR I and Hind III double digestion (5474bp+1135bp) product.
Fig. 5. be recombiant plasmid pYA3149-cagA 160-vacA 62-ureB 138Enzyme action identify agarose gel electrophoresis figure.
Wherein swimming lane 1 is that (D518A), swimming lane 2 is recombiant plasmid pYA3149-cagA to nucleic acid (DNA) molecular weight standard for TaKaRa, Wide Range DNA Marker (100～6,000) 160-vacA 62-ureB 138EcoR I and Hind III double digestion (3681bp+1135bp) product.
Fig. 6. be SDS-PAGE electrophoresis detection Recombinant Protein Expression.
Wherein swimming lane 1 be protein molecular weight standard (Fermentas, #SM0671), swimming lane 2 is induced the 24h contrast for empty carrier bacterium (χ 4550 (pYA3149)), swimming lane 3 is recombinant attenuated Salmonella typhi vaccine strain χ 4550 (pYA3149-cagA 160-vacA 62-ureB 138) to induce 24h, swimming lane 4 be recombinant attenuated Salmonella typhi vaccine strain χ 4550 (pYA3149-cagA 160-vacA 62-ureB 138) to induce 12h, swimming lane 5 be recombinant attenuated Salmonella typhi vaccine strain χ 4550 (pYA3149-cagA 160-vacA 62-ureB 138) to induce 8h, swimming lane 6 be recombinant attenuated Salmonella typhi vaccine strain χ 4550 (pYA3149-cagA 160-vacA 62-ureB 138) induce 4h.
Fig. 7. detect the immunoreactivity of recombiant protein for Western.
Wherein swimming lane 1 be protein molecular weight standard (Fermentas, #SM0671), swimming lane 2 is induced the 24h contrast for empty carrier bacterium (χ 4550 (pYA3149)), swimming lane 3 is recombinant attenuated Salmonella typhi vaccine strain χ 4550 (pYA3149-cagA 160-vacA 62-ureB 138) induce 24h.
Fig. 8. be reorganization attenuated salmonella typhimurium strain live vaccine oral immunity BALB/c mouse, ELISA method detection immunity back Hp specific serum IgG and gastric mucosa IgA antibody vertical coordinate are as a result represented 450nm place light absorption value.
Fig. 9 fusion gene cagA 160-vacA 62-ureB 138Make up sketch map
Figure 10 pYA3149-cagA 160-vacA 62-ureB 138The plasmid construction sketch map
The specific embodiment
Embodiment 1 obtains HpDNA fragment cagA 160, vacA 62And ureB 138
1. design of primers reaches synthetic
Hp 26695 genomes (NC_000915) amplifying nucleic acid sequential design primer with the GenBank announcement, forward primer 5 ' end is introduced EcoR I restriction enzyme site, downstream primer 5 ' end is introduced Hind III restriction enzyme site, and middle primer is introduced flexible Linker (GGGSGGGS) sequence (Fig. 9).
Design of primers is as follows:
Figure GSA00000053969900091
Primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.
2.PCR amplification target DNA
1) preparation of template
With " bacterial genomes DNA extraction test kit " (TIANGEN, DP302) extracting Hp 26695 is (available from the American Type Culture Collection of Unite States Standard type culture collection institute, ATCC, there is preservation in our unit, can provide as demonstration test to the public and use) genome, the operation by specification carries out.
2) pcr amplification
With Hp 26695 genomic DNAs is template, uses cagA respectively 160P1 and cagA 160The P2 cagA that increases 160, vacA 62P1 and vacA 62The P2 vacA that increases 62, ureB 138P1 and ureB 138The P2 ureB that increases 138Adopt precious biological engineering (Dalian) company limited (RaKaRa) high-fidelity DNA polymerase ( HS DNA Polymerase Code:DR010A) carries out pcr amplification, and reaction system is as follows:
Figure GSA00000053969900102
The PCR reaction condition is: 94 ℃ of pre-degeneration 5min; 94 ℃ of degeneration 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; 72 ℃ are extended 10min fully.Get 3 μ l PCR product after reaction finishes, 2.0% agarose gel electrophoresis detects (Fig. 1).Behind the agarose gel electrophoresis, reclaim the purpose fragment, obtain cagA 160, vacA 62And ureB 138Dna fragmentation.
Embodiment 2 makes up cagA 160-vacA 62-ureB 138Fusion gene
1. overlap extension PCR obtains cagA 160-vacA 62Fusion gene
With the cagA that reclaims 160And vacA 62Genetic fragment is a template, cagA 160P1 and vacA 62P2 is that primer carries out pcr amplification, and reaction system is as follows:
Figure GSA00000053969900111
The PCR reaction condition is: 94 ℃ of pre-degeneration 5min; 94 ℃ of degeneration 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, 10 circulations; Add cagA 160P1 (10 μ M) and vacA 62Each 2 μ l of P2 (10 μ M); 94 ℃ of degeneration 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 10min fully.Get 3 μ l PCR product after reaction finishes, 2.0% agarose gel electrophoresis detects.Show among Fig. 2: the fusion gene clip size is consistent with expectation, shows that overlap extension obtains fusion gene.Behind the agarose gel electrophoresis, reclaim the purpose fragment, obtain cagA 160-vacA 62Fusion gene.
2. overlap extension PCR obtains cagA 160-vacA 62-ureB 138Fusion gene:
With the cagA that reclaims 160-vacA 62Fusion gene and ureB 138Genetic fragment is a template, cagA 160P1 and ureB 138P2 is that primer carries out pcr amplification, and reaction system is as follows:
Figure GSA00000053969900112
The PCR reaction condition is: 94 ℃ of pre-degeneration 5min; 94 ℃ of degeneration 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, 10 circulations; Add cagA 160P1 (10 μ M) and ureB 138Each 2 μ l of P2 (10 μ M); 94 ℃ of degeneration 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 10min fully.Get 3 μ l PCR product after reaction finishes, 2.0% agarose gel electrophoresis detects.Show among Fig. 3: the fusion gene clip size is consistent with expectation, shows that overlap extension obtains fusion gene.Behind the agarose gel electrophoresis, reclaim the purpose fragment, obtain cagA 160-vacA 62-ureB 138Fusion gene.
Embodiment 3 recombiant plasmid pET 22b (+)-cagA 160-vacA 62-ureB 138Structure
1. enzyme action
With the cagA that obtains 160-vacA 62-ureB 138Fusion gene and pET 22b (+) plasmid use precious biological engineering (Dalian) company limited restricted enzyme EcoR I and Hind III to do double digestion respectively, to specifications operation.The enzyme action qualification result as shown in Figure 4.Agarose gel electrophoresis reclaims 5.5kb carrier segments and 1.1kb cagA respectively 160-vacA 62-ureB 138The fusion gene fragment.
2. connect
Concentration by UV spectrophotometer measuring target DNA fragment and carrier segments, be generally 1: 2～10 principle according to external source fragment and carrier mole ratio, carry out coupled reaction (using precious biological engineering (Dalian) company limited to connect test kit), the coupled reaction system is as follows:
CagA 160-vacA 62-ureB 138Enzyme action reclaims product 4 μ l; PET 22b (+) plasmid vector 1 μ l; Connect solution 5 μ l.Cumulative volume 10 μ l, 16 ℃ of coupled reactions 2 hours.
3. transform
To connect product, Transformed E coli DH5a, ammonia benzyl resistance screening positive recombinant.
4. evaluation recombiant plasmid
Single bacterium colony on the picking ammonia benzyl resistance screening flat board, the extracting plasmid is done double digestion with EcoR I and Hind III and is identified, identifies correct back recovery genetic fragment (Fig. 4).Plasmid is served the sea and is given birth to the order-checking of worker Bioisystech Co., Ltd, the protein sequence of fusion gene slice encode and expectation (seeing sequence table) in full accord.
Embodiment 4 recombiant plasmid pYA3149-cagA 160-vacA 62-ureB 138Structure and expression and the antigenicity of fusion rotein in recombinant attenuated salmonella detect
1. plasmid extraction
Extracting plasmid pYA3149 (Roy Curtiss doctor III of U.S. Washington university is so kind as to give, and there is preservation in our unit, can provide as demonstration test to the public and use) and pET 22b (+)-cagA 160-vacA 62-ureB 138
2. endonuclease reaction
Carrier pYA3149 and pET 22b (+)-cagA 160-vacA 62-ureB 138P all uses EcoR I and Hind III double digestion.Agarose gel electrophoresis reclaims back enzyme action purpose fragment.
3. coupled reaction
Concentration by UV spectrophotometer measuring target DNA fragment and carrier segments, be generally 1: 2～10 principle according to external source fragment and carrier mole ratio, carry out coupled reaction (using precious biological engineering (Dalian) company limited to connect test kit), the coupled reaction system is as follows:
CagA 160-vacA 62-ureB 138Enzyme action reclaims product 4 μ l; PYA3149 plasmid vector 1 μ l; Connect solution 5 μ l.Cumulative volume 10 μ l, 16 ℃ of coupled reactions obtained pYA3149-cagA in 2 hours 160-vacA 62-ureB 138(see figure 3).
4. transform
To connect product, electricity changes attenuated salmonella typhimurium strain strain χ 4550, and (Roy Curtiss doctor III of U.S. Washington university is so kind as to give, there is preservation in our unit, can provide as demonstration test to the public and use), electricity commentaries on classics condition is: voltage 2500V, electric capacity 25uF, resistance 200 Ω, electricity revolving cup 2mm, discharge time 3～5ms.
LB plate screening positive recombinant.
5. evaluation recombinant expression plasmid
Single bacterium colony on the picking LB flat board, the extracting plasmid is done double digestion with EcoR I and Hind III and is identified, identifies correct (Fig. 5).Plasmid pYA3149-cagA 160-vacA 62-ureB 138Successfully construct (Figure 10), and obtain recombinant attenuated Salmonella typhi vaccine strain χ 4550 (pYA3149-cagA 160-vacA 62-ureB 138).
6. express and identify
1) recombination engineering induces
Get and identify that correct recombination engineering is inoculated in the 5ml LB culture fluid, 37 ℃ of shaking tables are cultivated (200r/min) and are spent the night.Next day with the recombination engineering of incubated overnight in 1% ratio transferred species in 20ml LB culture fluid, 37 ℃ of shaking tables are cultivated (200r/min) when treating OD600=0.8, adding IPTG is that 1mmol/L induces to final concentration, in inducing 24 hours SDS-PAGE to detect Recombinant Protein Expression, do empty carrier bacterium (χ 4550 (pYA3149)) simultaneously and induce contrast.
2) SDS-PAGE detects Recombinant Protein Expression
Get the bacterium liquid that 1ml induced 24 hours, centrifugal, the resuspended precipitation of 100 μ l distilled waters adds 100 μ l 2x SDS-PAGE sample-loading buffers, mixing, boiling water bath 10 minutes.SDS-PAGE electrophoresis (4% concentrates glue 10% separation gel), coomassie brilliant blue staining, (Fermentas, #SM0671) check analysis electrophoresis result have the protein expression (Fig. 6) that conforms to the recombination fusion protein theoretical molecular (41kd) of design with dying Marker in advance.
7. antigenicity detects
1) Western detects the immunoreactivity of recombiant protein
With the immunoreactivity of the full bacterium antiserum evaluation of the anti-Hp of rabbit recombiant protein, the result shows that recombiant protein and the full bacterium antiserum of the anti-Hp of rabbit have good immunoreactivity (Fig. 7).
2) zoopery detects the immunogenicity of recombiant protein
With 2.0 * 10 8The recombinant attenuated Salmonella typhimurtum live vaccine of cfu (bacterium colony forms number) oral immunity BALB/c mouse, detect (Fig. 8 through ELISA, *: p＜0.01), (t check) analysed in credit by statistics, oral vaccine group and PBS (phosphate buffer, pH7.0) group compares, and all there were significant differences for immunity back Hp specific serum IgG and gastric mucosa IgA antibody, p＜0.01; Empty plasmid bacterium group is not remarkable with PBS group comparing difference, p＞0.05.
The result shows, this vaccine can induce serum IgG antibody and the gastric mucosa secretory IgA antibody that BALB/c mouse produces the high anti-Hp that tires, and oral immunity equivalent empty plasmid bacterium (χ 4550 (pYA3149)) can not induce the antibody of anti-Hp, confirms that recombinant attenuated Salmonella typhimurtum live vaccine has good immunogenicity.
Embodiment 5 recombinant attenuated Salmonella typhimurtum live vaccine treatments and the experiment of prevention Hp infection animal
1. prevention Hp infection animal experiment
By the immunogenicity methods immune mouse of animal experiment detection recombiant protein, each is organized to irritate simultaneously in mice 4 weeks after immunity and feeds the Hp bacterium liquid for preparing.All laboratory animals 24 hours in advance are jejunitas, cut off the water supply, and irritate and feed bacterium liquid 0.2ml, about 2.0 * 10 for each every 8CfuHp bacterium liquid; Respectively once 6 hours at interval, art time filling was fed and was supplied drinking water in back 2 hours the upper and lower noon.Irritate to feed equivalent empty plasmid bacterium (χ 4550 (pYA3149)) respectively and PBS in contrast.
The 4th all laboratory animals are all put to death and collect specimen behind the counteracting toxic substances, put to death preceding 24 hours jejunitas water.Dissect mice, take out the Mus stomach, cut open, wash out the gastric residue gently, with half mucosa tissue coating Hp culture medium, trilinear method streak inoculation, little aerobic cultivation, the field planting situation (table 1) of mice Hp behind the observation Hp counteracting toxic substances with normal saline along greater gastric curvature.
The recombinant attenuated Salmonella typhimurtum live vaccine prevention of table 1 Hp infectious effect
Figure GSA00000053969900141
(chi-square criterion) analysed in credit by statistics, and vaccine group compares with empty plasmid bacterium group and PBS group, and there were significant differences for Hp infection animal number average, p＜0.01; Empty plasmid bacterium group is not remarkable with PBS group comparing difference, p＞0.05.
The result shows: vaccine immunity group mice gastric Hp infection rate significantly is lower than empty plasmid bacterium matched group and PBS matched group, and protective rate is 83.3%, and mice infected gastric Hp field planting amount significantly is lower than empty plasmid bacterium matched group and PBS matched group.
Conclusion: this recombinant attenuated Salmonella typhimurtum live vaccine oral immunity mice, compare with immune group not with the empty plasmid bacterial immunity, can produce the resistance of attacking, the effect that has prevention Hp to infect at the Hp viable bacteria.
2. treatment Hp infection animal experiment
By the method in the experiment of prevention Hp infection animal, set up Hp infecting mouse animal model.Infect 8 weeks of back, with 2.0 * 10 8The recombinant attenuated Salmonella typhimurtum live vaccine of cfu per os pours into the immunity of mice stomach.Irritate to feed respectively equivalent empty plasmid bacterium (χ 4550 (pYA3149)) and PBS (phosphate buffer, pH7.0) in contrast.Immune 6 weeks of back are by the method in the experiment of prevention Hp infection animal, the field planting situation (table 2) of mice Hp after the observation immunization therapy.
The recombinant attenuated Salmonella typhimurtum live vaccine treatment of table 2 Hp infectious effect
Figure GSA00000053969900151
(chi-square criterion) analysed in credit by statistics, and vaccine group and empty plasmid bacterium group and PBS group relatively do not detect after Hp number of animals and the treatment Hp field planting amount and obviously reduce the animal number average there were significant differences, p＜0.01 after the treatment; Empty plasmid bacterium group is not remarkable with PBS group comparing difference, p＞0.05.
The result shows: vaccine immunity group mice gastric Hp field planting amount all significantly descends than empty plasmid bacterium matched group and PBS matched group, and protective rate is 76.7%; Part mice gastric Hp even obtain removing, the Hp clearance rate is 43.3%.
Conclusion: this recombinant attenuated Salmonella typhimurtum live vaccine oral immunity Hp mice infected, compare with immune group not with the empty plasmid bacterial immunity, can produce the immunization therapy effect to the Hp viable bacteria of mice gastric field planting, the effect that have treatment np to infect.
Appendix
SEQUENCE?LISTING
＜110〉Military Medical Univ No.3, P.L.A
＜120〉the recombination fusion protein vaccine and the attenuated live vector vaccine of treatment and prevention helicobacter pylori infections
<130>P101001/DSJ/CQ
<160>14
<170>PatentIn?version?3.3
<210>1
<211>160
<212>PRT
＜213〉cagA160 aminoacid sequence
<400>1
Glu?Ala?Ile?Asn?Gln?Glu?Pro?Val?Pro?His?Val?Gln?Pro?Asp?Ile?Ala
1 5 10 15
Thr?Thr?Thr?Thr?Asp?Ile?Gln?Gly?Leu?Pro?Pro?Glu?Ala?Arg?Asp?Leu
20 25 30
Leu?Asp?Glu?Arg?Gly?Asn?Phe?Ser?Lys?Phe?Thr?Leu?Gly?Asp?Met?Glu
35 40 45
Met?Leu?Asp?Val?Glu?Gly?Val?Ala?Asp?Ile?Asp?Pro?Asn?Tyr?Lys?Phe
50 55 60
Asn?Gln?Leu?Leu?Ile?His?Asn?Asn?Ala?Leu?Ser?Ser?Val?Leu?Met?Gly
65 70 75 80
Ser?His?Asn?Gly?Ile?Glu?Pro?Glu?Lys?Val?Ser?Leu?Leu?Tyr?Ala?Gly
85 90 95
Asn?Gly?Gly?Phe?Gly?Asp?Lys?His?Asp?Trp?Asn?Ala?Thr?Val?Gly?Tyr
100 105 110
Lys?Asp?Gln?Gln?Gly?Asn?Asn?Val?Ala?Thr?Leu?Ile?Asn?Val?His?Met
115 120 125
Lys?Asn?Gly?Ser?Gly?Leu?Val?Ile?Ala?Gly?Gly?Glu?Lys?Gly?Ile?Asn
130 135 140
Asn?Pro?Ser?Phe?Tyr?Leu?Tyr?Lys?Glu?Asp?Gln?Leu?Thr?Gly?Ser?Gln
145 150 155 160
<210>2
<211>62
<212>PRT
＜213〉vacA62 aminoacid sequence
<400>2
Leu?Gly?Thr?Asn?Ser?Ile?Ser?Asn?Val?Asn?Leu?Ile?Glu?Gln?Phe?Lys
1 5 10 15
Glu?Arg?Leu?Ala?Leu?Tyr?Asn?Asn?Asn?Asn?Arg?Met?Asp?Ile?Cys?Val
20 25 30
Val?Arg?Asn?Thr?Asp?Asp?Ile?Lys?Ala?Cys?Gly?Thr?Ala?Ile?Gly?Asn
35 40 45
Gln?Ser?Met?Val?Asn?Asn?Pro?Asp?Asn?Tyr?Lys?Tyr?Leu?Ile
50 55 60
<210>3
<211>138
<212>PRT
＜213〉ureB138 aminoacid sequence
<400>3
Asp?Thr?Leu?Asn?Glu?Ala?Gly?Cys?Val?Glu?Asp?Thr?Met?Ala?Ala?Ile
1 5 10 15
Ala?Gly?Arg?Thr?Met?His?Thr?Phe?His?Thr?Glu?Gly?Ala?Gly?Gly?Gly
20 25 30
His?Ala?Pro?Asp?Ile?Ile?Lys?Val?Ala?Gly?Glu?His?Asn?Ile?Leu?Pro
35 40 45
Ala?Ser?Thr?Asn?Pro?Thr?Ile?Pro?Phe?Thr?Val?Asn?Thr?Glu?Ala?Glu
50 55 60
His?Met?Asp?Met?Leu?Met?Val?Cys?His?His?Leu?Asp?Lys?Ser?Ile?Lys
65 70 75 80
Glu?Asp?Val?Gln?Phe?Ala?Asp?Ser?Arg?Ile?Arg?Pro?Gln?Thr?Ile?Ala
85 90 95
Ala?Glu?Asp?Thr?Leu?His?Asp?Met?Gly?Ile?Phe?Ser?Ile?Thr?Ser?Ser
100 105 110
Asp?Ser?Gln?Ala?Met?Gly?Arg?Val?Gly?Glu?Val?Ile?Thr?Arg?Thr?Trp
115 120 125
Gln?Thr?Ala?Asp?Lys?Asn?Lys?Lys?Glu?Phe
130 135
<210>4
<211>380
<212>PRT
＜213〉recombination fusion protein CagAl60-VacA62-UreB138 aminoacid sequence
<400>4
Met?Pro?Glu?Phe?Glu?Ala?Ile?Asn?Gln?Glu?Pro?Val?Pro?His?Val?Gln
1 5 10 15
Pro?Asp?Ile?Ala?Thr?Thr?Thr?Thr?Asp?Ile?Gln?Gly?Leu?Pro?Pro?Glu
20 25 30
Ala?Arg?Asp?Leu?Leu?Asp?Glu?Arg?Gly?Asn?Phe?Ser?Lys?Phe?Thr?Leu
35 40 45
Gly?Asp?Met?Glu?Met?Leu?Asp?Val?Glu?Gly?Val?Ala?Asp?Ile?Asp?Pro
50 55 60
Asn?Tyr?Lys?Phe?Asn?Gln?Leu?Leu?Ile?His?Asn?Asn?Ala?Leu?Ser?Ser
65 70 75 80
Val?Leu?Met?Gly?Ser?His?Asn?Gly?Ile?Glu?Pro?Glu?Lys?Val?Ser?Leu
85 90 95
Leu?Tyr?Ala?Gly?Asn?Gly?Gly?Phe?Gly?Asp?Lys?His?Asp?Trp?Asn?Ala
100 105 110
Thr?Val?Gly?Tyr?Lys?Asp?Gln?Gln?Gly?Asn?Asn?Val?Ala?Thr?Leu?Ile
115 120 125
Asn?Val?His?Met?Lys?Asn?Gly?Ser?Gly?Leu?Val?Ile?Ala?Gly?Gly?Glu
130 135 140
Lys?Gly?Ile?Asn?Asn?Pro?Ser?Phe?Tyr?Leu?Tyr?Lys?Glu?Asp?Gln?Leu
145 150 155 160
Thr?Gly?Ser?Gln?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Ser?Leu?Gly?Thr?Asn
165 170 175
Ser?Ile?Ser?Asn?Val?Asn?Leu?Ile?Glu?Gln?Phe?Lys?Glu?Arg?Leu?Ala
180 185 190
Leu?Tyr?Asn?Asn?Asn?Asn?Arg?Met?Asp?Ile?Cys?Val?Val?Arg?Asn?Thr
195 200 205
Asp?Asp?Ile?Lys?Ala?Cys?Gly?Thr?Ala?Ile?Gly?Asn?Gln?Ser?Met?Val
210 215 220
Asn?Asn?Pro?Asp?Asn?Tyr?Lys?Tyr?Leu?Ile?Gly?Gly?Gly?Ser?Gly?Gly
225 230 235 240
Gly?Ser?Asp?Thr?Leu?Asn?Glu?Ala?Gly?Cys?Val?Glu?Asp?Thr?Met?Ala
245 250 255
Ala?Ile?Ala?Gly?Arg?Thr?Met?His?Thr?Phe?His?Thr?Glu?Gly?Ala?Gly
260 265 270
Gly?Gly?His?Ala?Pro?Asp?Ile?Ile?Lys?Val?Ala?Gly?Glu?His?Asn?Ile
275 280 285
Leu?Pro?Ala?Ser?Thr?Asn?Pro?Thr?Ile?Pro?Phe?Thr?Val?Asn?Thr?Glu
290 295 300
Ala?Glu?His?Met?Asp?Met?Leu?Met?Val?Cys?His?His?Leu?Asp?Lys?Ser
305 310 315 320
Ile?Lys?Glu?Asp?Val?Gln?Phe?Ala?Asp?Ser?Arg?Ile?Arg?Pro?Gln?Thr
325 330 335
Ile?Ala?Ala?Glu?Asp?Thr?Leu?His?Asp?Met?Gly?Ile?Phe?Ser?Ile?Thr
340 345 350
Ser?Ser?Asp?Ser?Gln?Ala?Met?Gly?Arg?Val?Gly?Glu?Val?Ile?Thr?Arg
355 360 365
Thr?Trp?Gln?Thr?Ala?Asp?Lys?Asn?Lys?Lys?Glu?Phe
370 375 380
<210>5
<211>1141
<212>DNA
＜213〉cagA160-vacA62-ureB138 nucleotide sequence
<400>5
gaattcgaag?caatcaatca?agaaccagtt?ccccatgtcc?aaccagatat?agccactact 60
accaccgaca?tacaaggctt?accgcctgaa?gctagggatt?tacttgatga?aaggggtaat 120
ttttctaaat?tcactcttgg?cgatatggaa?atgttagatg?ttgagggagt?cgctgacatt 180
gatcctaatt?acaagttcaa?tcaattattg?attcacaata?acgctctgtc?ttctgtgtta 240
atggggagtc?ataatggcat?agaacctgaa?aaagtttcat?tattgtatgc?gggcaatggt 300
ggttttggag?acaaacacga?ttggaacgcc?accgttggtt?ataaagacca?acaaggtaac 360
aatgtggcta?cactcattaa?tgtgcatatg?aaaaacggca?gtggcttagt?catagcaggt 420
ggtgagaaag?ggattaataa?ccctagtttt?tatctctaca?aagaagacca?actcacaggc 480
tcacaaggag?gcggaagtgg?aggaggtagc?ttaggcacta?acagcattag?taatgttaat 540
ctaatagagc?aattcaaaga?gcgcctagcc?ctttacaaca?acaataaccg?catggatatt 600
tgtgtggtgc?gaaatactga?tgacattaaa?gcatgcggga?cggctatcgg?caatcaaagc 660
atggtgaata?accccgacaa?ttacaagtat?cttatcggag?gcggaagtgg?aggaggtagc 720
gacactttga?atgaagctgg?ttgtgtggaa?gacactatgg?cagctattgc?cggacgcact 780
atgcacactt?tccacactga?aggtgctggc?ggcggacacg?ctcctgatat?tattaaagta 840
gctggtgaac?acaacattct?tcccgcttcc?actaacccca?ctatcccttt?cactgtgaat 900
acagaagcag?aacacatgga?catgcttatg?gtgtgccacc?acttggataa?aagcattaaa 960
gaagatgttc?agttcgctga?ttcaaggatc?cgccctcaaa?ccattgcggc?tgaagacact?1020
ttgcatgaca?tggggatttt?ctcaatcacc?agctctgact?ctcaagctat?gggtcgtgtg?1080
ggtgaagtta?tcactagaac?ttggcaaaca?gctgacaaaa?acaagaaaga?attttaagct?1140
t 1141
<210>6
<211>28
<212>DNA
<213>cagAl60?P1
<400>6
gcggaattcg?aaacgctcaa?tcaagaac 28
<210>7
<211>48
<212>DNA
<213>cagA160?P2
<400>7
gctacctcct?ccacttccgc?ctccttgtga?gcctgtgagt?tggtcttc 48
<210>8
<211>50
<212>DNA
<213>vacA62?P1
<400>8
ggaggcggaa?gtggaggagg?tagcttaggc?actaacagca?ttagtaatgt 50
<210>9
<211>50
<212>DNA
<213>vacA62?P2
<400>9
gctacctcct?ccacttccgc?ctccgataag?atacttgtaa?ttgtcggggt 50
<210>10
<211>49
<212>DNA
<213>ureB138?P1
<400>10
ggaggcggaa?gtggaggagg?tagcgacact?ttgaatgaag?ctggttgtg 49
<210>11
<211>33
<212>DNA
<213>ureB138?P2
<400>11
gcgaagctta?aaattctttc?ttgtttttgt?cag 33
<210>12
<211>480
<212>DNA
＜213〉cagA160 nucleotide sequence
<400>12
gaagcaatca?atcaagaacc?agttccccat?gtccaaccag?atatagccac?tactaccacc 60
gacatacaag?gcttaccgcc?tgaagctagg?gatttacttg?atgaaagggg?taatttttct 120
aaattcactc?ttggcgatat?ggaaatgtta?gatgttgagg?gagtcgctga?cattgatcct 180
aattacaagt?tcaatcaatt?attgattcac?aataacgctc?tgtcttctgt?gttaatgggg 240
agtcataatg?gcatagaacc?tgaaaaagtt?tcattattgt?atgcgggcaa?tggtggtttt 300
ggagacaaac?acgattggaa?cgccaccgtt?ggttataaag?accaacaagg?taacaatgtg 360
gctacactca?ttaatgtgca?tatgaaaaac?ggcagtggct?tagtcatagc?aggtggtgag 420
aaagggatta?ataaccctag?tttttatctc?tacaaagaag?accaactcac?aggctcacaa 480
<210>13
<211>186
<212>DNA
＜213〉vaca62 nucleotide sequence
<400>13
ttaggcacta?acagcattag?taatgttaat?ctaatagagc?aattcaaaga?gcgcctagcc 60
ctttacaaca?acaataaccg?catggatatt?tgtgtggtgc?gaaatactga?tgacattaaa 120
gcatgcggga?cggctatcgg?caatcaaagc?atggtgaata?accccgacaa?ttacaagtat 180
cttatc 186
<210>14
<211>414
<212>DNA
＜213〉ureB138 nucleotide sequence
<400>14
gacactttga?atgaagctgg?ttgtgtggaa?gacactatgg?cagctattgc?cggacgcact 60
atgcacactt?tccacactga?aggtgctggc?ggcggacacg?ctcctgatat?tattaaagta 120
gctggtgaac?acaacattct?tcccgcttcc?actaacccca?ctatcccttt?cactgtgaat 180
acagaagcag?aacacatgga?catgcttatg?gtgtgccacc?acttggataa?aagcattaaa 240
gaagatgttc?agttcgctga?ttcaaggatc?cgccctcaaa?ccattgcggc?tgaagacact 300
ttgcatgaca?tggggatttt?ctcaatcacc?agctctgact?ctcaagctat?gggtcgtgtg 360
ggtgaagtta?tcactagaac?ttggcaaaca?gctgacaaaa?acaagaaaga?attt 414

Claims (10)

  1. One kind the treatment and the prevention helicobacter pylori infections the recombination fusion protein vaccine; comprise one section recombination fusion protein, it is characterized in that described recombination fusion protein origin comes from helicobacter pylori cytotoxin related gene PROTEIN C agA, the vacuolating cytotoxin VacA of helicobacter pylori and the immunity protection function fragment linearity that has of urease subunit UreB connects and composes.
  2. 2. recombination fusion protein vaccine according to claim 1, the immunity protection function fragment CagA of described helicobacter pylori cytotoxin related gene PROTEIN C agA 160Aminoacid sequence shown in Seq ID No.1; The immunity protection function fragment VacA of described vacuolating cytotoxin VacA 62Aminoacid sequence shown in Seq ID No.2; The immunity protection function fragment UreB of described urease subunit UreB 138Aminoacid sequence shown in Seq ID No.3.
  3. 3. recombination fusion protein vaccine according to claim 2 has CagA 160-VacA 62-UreB 138The order of connection.
  4. 4. according to claim 2 or 3 described recombination fusion protein vaccines, described CagA 160, VacA 62, UreB 138Between adopt connection peptides.
  5. 5. recombination fusion protein vaccine according to claim 4, described recombination fusion protein have the aminoacid sequence shown in Seq ID No.4.
    6 one kinds of recombiant plasmid comprise skeleton carrier and exogenous gene, and described exogenous gene comprises the gene of the arbitrary described recombination fusion protein vaccine of coding claim 1～5.
  6. 7. recombiant plasmid according to claim 6, described skeleton carrier is for containing the segmental Salmonella typhimurium bacteria plasmid of aspartic acid β semialdehyde dehydrogenase gene pYA3149.
  7. One kind the treatment and the prevention helicobacter pylori infections the attenuated live vector vaccine, comprise carrier bacterium and express immunogenic exogenous plasmid, described carrier bacterium is an attenuated live, described exogenous plasmid is above-mentioned recombiant plasmid, described attenuated live refer to by behind the gene engineering method attenuation to host's pathogenic remarkable reduction, and kept bacterial strain to the infection ability of the mucosa of stomach, intestinal or cell.
  8. 9. attenuated live vector vaccine according to claim 8, described attenuated live are the attenuated salmonella typhimurium strain strain.
  9. 10. attenuated live vector vaccine according to claim 9, described attenuated salmonella typhimurium strain strain are the bacterial strain χ 4550 that has lacked aspartic acid β semialdehyde dehydrogenase gene.
  10. 11. the preparation method of the described attenuated live vector vaccine of claim 10, step is as follows:
    (1) with the helicobacter pylori genome be template, amplification has the genetic fragment of nucleotide sequence shown in Seq ID No.12, Seq ID No.13, the Seq ID No.14 respectively.
    (2) ligation amplification obtains three genetic fragments, and is building up to and contains on the segmental skeleton carrier pYA3149 of aspartic acid β semialdehyde dehydrogenase gene, the screening recombiant plasmid;
    (3) recombiant plasmid changes among the host bacterium χ 4550,
    The primer of the genetic fragment of nucleotide sequence is as follows respectively shown in amplification Seq ID No.12, Seq ID No.13, the Seq ID No.14:
    cagA 160?P1/cagA 160?P2：
    5-GCG
    Figure FSA00000053969800021
    GAAACGCTCAATCAAGAAC-3，
    5-GCTACCTCCTCCACTTCCGCCTCCTTGTGAGCCTGTGAGTTGGTCTTC-3；
    vacA 62?P1/vacA 62?P2：
    5GGAGGCGGAAGTGGAGGAGGTAGCTTAGGCACTAACAGCATTAGTAATGT3，
    5GCTACCTCCTCCACTTCCGCCTCCGATAAGATACTTGTAATTGTCGGGGT3；
    ureB 138?P1/ureB 138?P2：
    5GGAGGCGGAAGTGGAGGAGGTAGCGACACTTTGAATGAAGCTGGTTGTG3，
    5GCGAAGCTTAAAATTCTTTCTTGTTTTTGTCAG3。
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CN102636641A (en) * 2012-03-26 2012-08-15 上海凯创生物技术有限公司 Detection kit of helicobacter pylori emulsion method and preparation process thereof
CN102643850A (en) * 2012-04-18 2012-08-22 天津天佛罗生物技术有限公司 Preparation method of helicobacter pylori virulence proteome
CN110846335A (en) * 2018-12-22 2020-02-28 波音特生物科技（南京）有限公司 Preparation method of antibody kit capable of detecting highly pathogenic strains of helicobacter pylori
CN112107682A (en) * 2020-10-13 2020-12-22 宁夏医科大学 M cell targeting recombinant lactobacillus vaccine, preparation method and application
CN112143750A (en) * 2020-10-13 2020-12-29 宁夏医科大学 M-cell-targeted recombinant lactobacillus surface display system, recombinant lactobacillus vaccine, preparation method and application
CN112190704A (en) * 2020-10-13 2021-01-08 宁夏医科大学 M cell targeting recombinant lactobacillus vaccine, preparation method and application
CN112402600A (en) * 2020-10-13 2021-02-26 宁夏医科大学 Helicobacter pylori tetravalent virulence factor GEM particle vaccine, preparation method and application
CN113846080A (en) * 2021-09-06 2021-12-28 尤丽康（江苏）生物医药有限公司 Helicobacter pylori composite antigen, helicobacter pylori antibody, preparation method and application
CN115724922A (en) * 2022-07-19 2023-03-03 四川大学华西医院 Helicobacter pylori vaccine recombinant protein antigen TonB, and preparation method and application thereof
CN116903711A (en) * 2023-06-07 2023-10-20 四川大学华西医院 Helicobacter pylori vaccine recombinant protein antigen CagA1, preparation method and application thereof
CN116970045A (en) * 2023-06-02 2023-10-31 四川大学华西医院 Helicobacter pylori vaccine recombinant protein antigen MCP, preparation method and application thereof
EP4338749A1 (en) * 2022-09-14 2024-03-20 Iguana Biotechnology GmbH Vaccine against helicobacter pylori infection

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CN1593169A (en) * 2003-09-09 2005-03-16 上海立泰哲田生命科技有限公司 Specific anti-helicobacter pylori immunological cow foremilk preparation method and its uses
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CN102636641A (en) * 2012-03-26 2012-08-15 上海凯创生物技术有限公司 Detection kit of helicobacter pylori emulsion method and preparation process thereof
CN102643850A (en) * 2012-04-18 2012-08-22 天津天佛罗生物技术有限公司 Preparation method of helicobacter pylori virulence proteome
CN102643850B (en) * 2012-04-18 2013-08-21 天津天佛罗生物技术有限公司 Preparation method of helicobacter pylori virulence proteome
CN110846335A (en) * 2018-12-22 2020-02-28 波音特生物科技（南京）有限公司 Preparation method of antibody kit capable of detecting highly pathogenic strains of helicobacter pylori
CN110846335B (en) * 2018-12-22 2023-02-28 波音特生物科技（南京）有限公司 Preparation method of antibody kit capable of detecting highly pathogenic strains of helicobacter pylori
CN112402600B (en) * 2020-10-13 2022-11-18 宁夏医科大学 Helicobacter pylori tetravalent virulence factor GEM particle vaccine, preparation method and application
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CN112402600A (en) * 2020-10-13 2021-02-26 宁夏医科大学 Helicobacter pylori tetravalent virulence factor GEM particle vaccine, preparation method and application
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CN115724922A (en) * 2022-07-19 2023-03-03 四川大学华西医院 Helicobacter pylori vaccine recombinant protein antigen TonB, and preparation method and application thereof
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EP4338749A1 (en) * 2022-09-14 2024-03-20 Iguana Biotechnology GmbH Vaccine against helicobacter pylori infection
WO2024056825A1 (en) * 2022-09-14 2024-03-21 Iguana Biotechnology Gmbh Vaccine against helicobacter pylori infection
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CN116970045B (en) * 2023-06-02 2024-05-14 四川大学华西医院 Helicobacter pylori vaccine recombinant protein antigen MCP, preparation method and application thereof
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