CN1864746A - Gene engineered poly-valence subunit vaccine of Hp AhpC and preparation method thereof - Google Patents

Abstract

The disclosed vaccine employs alkyl hydrogen peroxide reductase (AhpC) or its fusion gene with neutral granular leukocyte activated protein NapA as active constituent, as well as intra-molecule external adjuvant such as bacillus coli heat-labile toxin B subunit LTB, cholera morbus toxin B subunit CTB.

Description

Gene engineered poly-valence subunit vaccine of Hp AhpC and preparation method thereof

Technical field

The invention belongs to field of biological pharmacy, relate to the recombinant vaccine and the multivalent subunit vaccine preparation method thereof that are used for immunity of people's helicobacter pylori infections and treatment.

Background technology

Helicobacter pylori (helicobacter pylori, H.pylori) be the gram negative bacilli that is colonizated in people's gastric mucosa and causes stomach, dudenal disease, the about population stomach infections helicobacter pylori more than 50% in the whole world, and obtain in the Childhood mostly, as not treating with persistent infection, cause chronic gastritis, peptic ulcer, even cause gastric cancer.Since nineteen eighty-three Warren and Marshall discovery helicobacter pylori, people are devoted to the research of this antibacterial always, World Health Organization (WHO) and NIH formally classify H.pylori as the primary cancerigenic factor of gastric cancer through for many years analysis and research.Now clinical many triple therapies, quadruple chemotherapy treatment with proton pump inhibitor added with antibiotic or bismuth salt, this therapeutic scheme has certain effect but also must be in the face of some problem that cause, such as medical expense costliness, erious adverse reaction, patient's compliance and antibiotic drug resistance with infect once more etc.Therefore develop a kind of effective vaccine and may be the effective way of digestive tract disease due to prevention and the treatment helicobacter pylori infections.

But Hp yeast culture condition harshness is difficult to carry out large-scale culture.Bacterial antigens component complexity, and content is lower, directly from full bacterium separation and purification to go out the difficulty of protective antigen bigger, method is loaded down with trivial details, is unfavorable for the industrialization preparation of vaccine.Utilize technique for gene engineering that the effective protective antigen of thalline is carried out clonal expression the feasibility of Hp vaccine development is improved greatly, wherein genetic engineering urease subunit vaccine has entered the III clinical trial phase stage.Meanwhile, many subunits amalgamation protein vaccine also more and more is subjected to people's attention.Ruggiero etc. think in the bacterial infection process; because complicated mechanism of action is arranged between host and the pathogenic bacterium; the vaccine that single antigen component makes up is difficult to produce protective effect in full force and effect; on the single protective antigen vaccine research of Hp basis; the many subunit vaccines that make up have many antigen components of Hp; can produce than the stronger immunoreation of single subunit composition by excitating organism, antigenic component is more simple with respect to whole-bacterial-vaccine, can reduce the allergic generation of body.

The gram negative bacteria outer membrane protein has good antigen active; these outer membrane protein are as the main target of antibody and immunocyte attack; can mediate the most direct effective killing action of antibacterial, be whether the decision immunoreation has protectiveness to human body key factor.Recently studies have shown that in all inflammatory effects factors of Hp, oxidative stress is the important virulence factor that causes the stomach inflammation.The inductive oxidative stress of Hp can cause the damage of host cell, also will face the attack of the oxygen-derived free radicals of host's phagocyte release simultaneously.Hp must have a kind of protection mechanism defence oxidative stress, and alkyl peroxide reductase (AhpC) is exactly a kind of important antioxidase, the damage that prevention Hp brings under oxygen impacts.

It is feature with neutrophilic granulocyte and monocyte infiltration that gastric mucosa infects Hp, closely related (the Bayerdorffer E of its extent of damage and neutrophil infiltration, Lehn N, Hatz R, et al.Difference in expressionof Helicobacter pylori gastritis inantrum and body.Gastroen-terology, 1992,102:1575-1582).Discover have one or more protein ingredients to have the effect of direct attraction and activation neutrophilic granulocyte and other inflammatory cells in the Hp water extract in early days.The relative molecular mass of purifying thus is 15KDa, have and promote the neutrophil adhesion endotheliocyte and induce neutrophilic granulocyte to produce the material of reactive oxygen free radical, called after NAP (Evans DJ Jr, Evans DG, Takemura T, et al.Characterization of aHelicobacter pylorineutrophil activating protein.Infect Immun, 1995,63:2213-2220).HP-NAP is positioned at the Hp thalline, after self-dissolving release, be incorporated into bacterium surface, play the effect of adhesin, mediation and mucinous combination the (Namavar F, Sparrius M, Veerman EC, et al.Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfatedcarbohydrates on high molecular weight salivary mucin.Infect Immun, 1998,66:444-447.), perhaps combine with the polymorphonuclear leukocyte sphingomyelin.Also can promote the secretion of the synthetic and plasminogen activator inhibitor-2 of MCTF.Coordinate expression by inducing cell procoagulant and anti-fibrinolytic, HP-NAP can promote fibrin deposition and gastric mucosa inflammation reaction (the Montemurro P that is caused by Hp, Barbuti G, Dundon WG, et al.Helicobacter pylori neutrophilactivating protein stimulates tissue factor and plas minogen activator inhibitor production by human bloodmononu clear cells.J Infect Dis, 2001,183:1055-1062.).

AhpC and NapA are the outer membrane protein compositions of Hp, and its encoding gene has high conservative, are the important candidate antigens of Hp vaccine.Have not yet to see and use helicobacter pylori envelope antigen AhpC, particularly use the report of the fusion rotein of AhpC and NapA as immunogenicity material preparation helicobacter pylori polyvalent vaccine as the immunogenicity material.

Summary of the invention

Purpose of the present invention aims to provide Hp genetic engineering multivalent subunit vaccine of a kind of highly effective and safe and preparation method thereof at the deficiency that existing vaccine exists.

This vaccine adopt alkyl peroxide enzyme AhpC and with the fusion rotein of neutrophil activating protein NapA, obtain single recombiant protein and merge recombiant protein, again with the adjuvant composition, as E.coli LT B subunit (LTB), choleratoxin B subunit CTB or aluminum salt adjuvant etc., prepare the Hp recombinant vaccine of dissimilar (outer adjuvant of molecule or intramolecularly adjuvants) in various combination mode (mixing of albumen physical property or gene level fusion etc.).

In order to obtain helicobacter pylori AhpC recombinant vaccine of the present invention, can carry out the single-gene clone at Hp protective antigen composition alkyl peroxide enzyme AhpC, obtain single recombiant protein.With it as the primary activity composition, again with different compound modes and adjuvant composition, as E.coli LT B subunit LTB, choleratoxin B subunit CTB etc., carry out physical property with proper proportion to be mixed with out Hp multivalence combination-vaccine, be used for the mucosal route immunity inoculation.Animal test results shows: dissimilar vaccines all excite stronger systemic immunity and mucosal immunity after with the different vaccination ways immunity, obtain immune protective effect preferably.

Perhaps, alkyl peroxide enzyme AhpC, NapA are carried out gene level to be connected, the construction expression recombination fusion protein, and prepare the different molecular genetic engineering reorganization multivalence amalgamation protein vaccine of adjuvant outward with adjuvant compositions such as E.coli LT B subunit LTB, choleratoxin B subunit CTB and aluminum salt adjuvants according to the proper proportion physical mixed on this basis.This vaccine is applicable to the mucosal route immunity inoculation, can excite stronger mucosal immunity of generation and systemic immunity to reply, and reaches the purpose that immunoprophylaxis and treatment Hp infect.

Therefore, as the active component of helicobacter pylori AhpC recombinant vaccine of the present invention, it can be the single antigen component alkyl peroxide of a helicobacter pylori reductase (AhpC).Or with Heliobacter pylori antigen component AhpC and NapA by the joint sequence resulting fused protein that links together.Or the fusion gene of Heliobacter pylori antigen component AhpC or itself and NapA and adjuvant composition LTB or CTB are merged the intramolecularly adjuvant genetic engineering recombinant fusion protein of formation by joint sequence on gene level.

Can use method well known to those skilled in the art (referring to Sambrook, ed.Molecular Cloning:A Laboratory Manul (2nd.ed.), Vols.1-3, Cold Spring Harbor Laboratory (1998); Current Protocols In Molrcular Biology, Ausubel, ed.John wiley ﹠amp; Sons, Inc.New York (1997)), prepare the helicobacter pylori AhpC recombinant vaccine that is added with the intramolecularly adjuvant of the present invention.For example, can be under the connection of joint sequence with alkyl peroxide enzyme AhpC, NapA, on gene level, merge adjuvant composition E.coli LT B subunit (LTB) or choleratoxin B subunit CTB, the construction expression recombination fusion protein is prepared intramolecularly adjuvant multivalence amalgamation protein vaccine.Briefly, this method may further comprise the steps:

(1) provides pcr amplification, gene clone and the sequence analysis of helicobacter pylori AhpC, NapA protective antigen and E.coli LT B subunit LTB, choleratoxin B subunit CTB genes of interest;

(2) the helicobacter pylori AhpC of step (1) or itself and NapA, the thermo-labile intestinal poison of escherichia coli B subunit LTB, the full gene of choleratoxin B subunit CTB or function fragment separately are connected in prokaryotic expression plasmid or the polygenes fusion expression plasmid;

(3) recombinant expression plasmid that obtains with step (2) transforms suitable host bacteria cell, obtains gene process recombinant bacterial strain,

(4) be suitable for expressing under the condition of desired protein the genetic engineering recombination strain of large scale fermentation incubation step (3);

(5) separate the also recombinant protein of purification step (4) generation, promptly obtain helicobacter pylori AbpC recombinant vaccine.

According to a preferred embodiment of the invention, clone AhpC, NapA, LTB in the said step 1, the employed primer of CTB gene is respectively:

AhpC P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

P2 5’-CG CTCGAGAAGCTTAATGGAAT-3’

XhoI

P2’ 5’- GCTACCTCCGCCTCCAAGCTTAATGGAAT

Linker

NapA P3 5’- CATATGAAAACATTTGAAATT-3’

NdeI

P3’ 5’-T GGAGGCGGAGGTAGCAAAACATTTGAAATT-3

linker

P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

P4’ 5’- GCTACCTCCTCCACTTCCGCCTCCAGCCAAATGGGCTTCTAG-3’

linker

LTB P5 5’- CCATGGCTCCCCAGTCTATTAC-3’

NcoI

P5’ 5’- GGAGGCGGAAGTGGAGGAGGTAGCGCTCCCCAGTCTATT-3’

linker

P5” 5’- GGAGGCGGAGGTAGCGCTCCCCAGTCTATT-3’

linker

P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

CTB P7 5’- CATATGATTAAATTAAAATTTG-3’

NcoI

P7’ 5’- GGAGGCGGAAGTGGAGGAGGTAGCATTAAATTAAAATTTGGT-3’

linker

P7” 5’- GGAGGCGGAGGTAGCATTAAATTAAAATTTGGT-3’

linker

P8 5’ CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

According to a preferred embodiment in addition of the present invention, the employed synthetic primer of gene fusion construct is respectively in the said step 2:

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

NdeI

AhpC

P2 5’-CG CTCGAGTTAAAGCTTAATGGAAT-3’

XhoI

P3 5’- CATATGAAAACATTTGAAATT-3’

NdeI

NapA

P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

P5 5’- CCATGGCTCCCCAGTCTATTAC-3’

NcoI

LTB

P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

CTB

P7 5’- CATATGATTAAATTAAAATTTG-3’

NcoI

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-LTB

P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-CTB

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-NapA

P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-NapA-LTB

P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-NapA-CTB

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

According to another preferred embodiment of the present invention, the used anion filler of purified fusion protein is selected from Q Sepharose HP, Q Sepharose FF or Q Sepharose XL in the said step 4; Nickel ion affinity purification filler is selected from ChelatingSepharose HP or Chelating Sepharose FF; The gel permeation chromatography column packing is selected from Superdex 75, Superdex 200 or Superdex HR 10/30.

Vaccine of the present invention can carry out immunity inoculation by mucosal route (gastrointestinal mucosa, nasal mucosa etc.), and excitating organism produces high titre sIgA and stronger mucosal immune response and certain systemic immunity and replys.

Our animal test results shows that unit price of the present invention or polyvalent vaccine have good anti-Hp immunoprophylaxis effect that infects and the immunization therapy effect of removing the Hp infection.

Genetic engineering Recombinant Protein Expression of the present invention and fundamental characteristics: the 1. equal abduction delivering in prokaryotic expression system-escherichia coli of constructed recombinant expression plasmid among the present invention.2. the expression rate of recombinant protein A hpC is about 37%; Recombination fusion protein AhpC-LTB expression rate is about 34%, and recombination fusion protein AhpC-NapA-LTB expression rate is about 30%, and recombination fusion protein AhpC-CTB expression rate is about 31%, recombination fusion protein AhpC-NapA-CTB expression rate about 25%; 3. each recombiant protein is all with the inclusion body formal representation, and the purity behind the purification is greater than 80%.4. each recombiant protein all can induced animal produces the specific antibody of higher titre.

The present invention adopts technique for gene engineering clonal expression protective antigen composition, and the expression height is convenient to separation and purification, and highly effective and safe.This recombiant protein can directly combine with adjuvant (LTB, CTB), prepares outer adjuvant of multiple molecule or intramolecularly Adjuvanted vaccines, and form is versatile and flexible, is applicable to different methods of vaccination and approach.

Description of drawings

Fig. 1 shows genome extracting sepharose electrophoresis result.Nucleic acid (DNA) molecular weight standard (Marker); Swimming lane 2 is the Hp genomic DNA.

Fig. 2 shows ahpC gene PCR amplification.Swimming lane 1 is nucleic acid (DNA) molecular weight standard (Marker); Swimming lane 2 is the pcr amplification product (594bp) of genes of interest ahpC.The diagram result shows that the pcr amplification of genes of interest ahpC is successful.

Fig. 3 shows the qualification result of cutting of cloning vehicle pMD-18-ahpC.Swimming lane 1 is nucleic acid (DNA) molecular weight standard (Marker); The sub-enzyme action result of swimming lane 2 negative clones; The sub-enzyme action results of swimming lane 3,4 positive clones (594bp).Diagram is the result show, the ahpC genetic fragment size of enzyme action is consistent with expectation, shows the success of T carrier cloning.

Fig. 4 shows the qualification result of cutting of expression vector pET22b-ahpC.Swimming lane 1 is pET22b positive colony; Swimming lane 2 is nucleic acid (DNA) molecular weight standard (Marker).Diagram is the result show, the ahpC genetic fragment size of enzyme action is consistent with expectation, and the clone who shows expression vector is successful.

Fig. 5 shows that SDS-PAGE detects the rAhpC protein expression.Wherein swimming lane 1 is kind of a daughter bacteria; Swimming lane 2 is pET22b/BL21; Swimming lane 3 was induced 2 hours for reorganization pET22b-ahpC/BL21; Swimming lane 4 is induced 4 hours (h) for reorganization pET22b-ahpC/BL21; Swimming lane 5 is induced 6h for reorganization pET22b-ahpC/BL21; Swimming lane 6 is induced 8h for reorganization pET22b-ahpC/BL21; Swimming lane 7 is the molecular weight of albumen standard.Diagram is the result show, is 37.6% through UVP scanning analysis destination protein expression.

Fig. 6 shows the proteic expression-form of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) evaluation rAhpC.Wherein swimming lane 1 is a recombination engineering; Swimming lane 2 is the engineering bacteria ultrasound precipitation; Swimming lane 3 is the ultrasonic supernatant of engineering bacteria; Swimming lane 4 to 7 is the inclusion body washing; Swimming lane 8 is the AhpC inclusion body; Swimming lane 9 is the molecular weight of albumen standard.

Fig. 7 shows napA, ahpC, ltB gene PCR amplification.Wherein swimming lane 1 is nucleic acid molecular weight standard (Marker); Swimming lane 2 is the pcr amplification product (432bp) of genes of interest napA; Swimming lane 3 is the pcr amplification product (594bp) of genes of interest ahpC; Swimming lane 4 is the pcr amplification product (309bp) of genes of interest ltB.Illustrated result shows, genes of interest napA, and the PCR clonal expansion of ahpC and ltB is respond well.

Fig. 8 shows fusion gene ahpC-ltB overlap extension PCR electrophoresis result (918bp).Wherein swimming lane 1 is nucleic acid molecular weight standard (Marker); Swimming lane 2 is the pcr amplification product of fusion gene ahpC-ltB.By the diagram result as can be seen, the fusion gene clip size is consistent with expectation, shows that overlap extension obtains fusion gene.

Fig. 9 shows ahpC genetic fragment, napA-ltB fusion gene PCR electrophoresis result.Wherein swimming lane 1 is nucleic acid molecular weight standard (Marker); Swimming lane 2 is the pcr amplification product of genes of interest ahpC; Swimming lane 3 is the pcr amplification product of fusion gene napA-ltB.

Figure 10 shows fusion gene ahpC-napA-ltB overlap extension PCR electrophoresis result.Wherein swimming lane 1 is the pcr amplification product of fusion gene ahpC-napA-ltB; Swimming lane 2 is nucleic acid molecular weight standard (Marker).By the diagram result as can be seen, the fusion gene clip size is consistent with expectation, shows that overlap extension obtains fusion gene.

Figure 11 shows the qualification result of cutting of recombiant plasmid pMD18-T-ANL.Wherein swimming lane 1 is nucleic acid molecular weight standard (Marker); The sub-enzyme action result of swimming lane 2,4 negative clones; The sub-enzyme action result of swimming lane 3 positive clones.By the diagram result as can be seen, enzyme action genetic fragment size is consistent with theoretical value, shows the success of T carrier cloning.

Figure 12 shows the qualification result of cutting of recombiant plasmid pET22b-ANL.The sub-enzyme action result of swimming lane 1 positive clone wherein.Wherein swimming lane 2 is nucleic acid molecular weight standard (Marker).Show among the figure that enzyme action genetic fragment size is consistent with theoretical value, show that expression vector clones successfully.

Figure 13 shows the protein expression with SDS-PAGE method detection rANL.Wherein swimming lane 1 is kind of a daughter bacteria; Swimming lane 2 is pET22b/BL21; Swimming lane 3 was induced 2 hours for reorganization pET22b-ANL/BL21; Swimming lane 4 was induced 4 hours for reorganization pET22b-ANL/BL21; Swimming lane 5 was induced 6 hours for reorganization pET22b-ANL/BL21; Swimming lane 6 is the molecular weight of albumen standard.It is 32.7% that the result shows through UVP scanning analysis destination protein expression.

Figure 14 shows rANL expression-form identification experiment result.Wherein swimming lane 1 is a recombination engineering; Swimming lane 2 is the engineering bacteria ultrasound precipitation; Swimming lane 3 is the ultrasonic supernatant of engineering bacteria; Swimming lane 4-7 is the inclusion body washing; Swimming lane 8 is the rANL inclusion body; Swimming lane 9 is the molecular weight of albumen standard.

Figure 15 shows proteantigen Quality Identification figure (SDS-PAGE).Wherein swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is rAhpC; Swimming lane 3 is rANL.

The specific embodiment

Below in conjunction with drawings and Examples the present invention is described in detail the present invention.

The clone of the outer adjuvant LTB of embodiment 1 helicobacter pylori AhpC, NapA encoding gene and molecule, CTB gene

1. helicobacter pylori derives from Gastroenterology dept. of southwestern hospital patients clinical separated strain (strain number: CQ9803)

2. take out the Hp bacterial strain of preserving (CQ 9803) in the liquid nitrogen container and coat on the Hp special solid culture medium,, contain 5%O in 37 ℃ ₂, 85%N ₂, 10%CO ₂Cultivate 72h under the condition.Genome extraction agent box extracting Hp genome (shown in the accompanying drawing 1).

3. adopt PCR method from increase the respectively encoding gene of AhpC and NapA of Hp genome.

1) design of primers synthetic following (underscore shows restriction enzyme site and joint (linker) base sequence)

Gene order and design of primers principle according to GenBank announces design corresponding primer, introduce restriction enzyme site.Introduce following joint (linker) sequence in the centre.

P1 5’-CG CATATGTTAGTTACAAAACTTGC-3’

Nde I

AhpC

P2 5’-CG CTCGAGTTAAAGCTTAATGGAAT-3’

XhoI

NapA

P3 5’- CATATGAAAACATTTGAAATT-3’

NdeI

P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

P5 5’- CCATGGCTCCCCAGTCTATTAC-3’

NcoI

LTB

P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

P7 5’- CATATGATTAAATTAAAATTTG-3’

NcoI

CTB

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

2) pcr amplification of genes of interest:

With helicobacter pylori genome DNA is template, P1 and P2, P3 and P4 primer increase respectively AhpC, NapA gene, producing malicious escherichia coli H44815 genomic DNA with wild type is that template is with P5 and P6 primer amplification LTB gene, with vibrio cholera (El Tor biotype) genomic DNA is template, P7 and P8 primer amplification CTB gene, adopt following PCR system and program (the PCR test kit is available from Dalian TaKaRa company product):

Template DNA 1 μ l; 10 * PCR buffer (containing magnesium chloride), 5 μ l; DNTPs (10mmol/L) 4 μ l; Each 1 μ l of upstream and downstream primer (0.025mmol/L); Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l adds deionized water to final volume 50 μ l.

With the reaction system mixing, after the centrifugal treating, add 30 μ l paraffin oil.94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 40 seconds, 72 ℃ were extended 40 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Get 1 μ l product after reaction finishes, 1.5% agarose gel electrophoresis detects the PCR effect.

3) recovery of PCR product (reclaiming test kit is Time Inc. available from sky, Beijing, presses the operation of test kit operation instructions)

(1) records 1.0% agarose gel;

(2) pcr amplification product is added on the electrophoresis in the sample hole, stop electrophoresis when indicator migrates to the appropriate location;

(3) separation contains the segmental gel of purpose under Burdick lamp, moves into 1.5ml EP pipe;

(4) add DNA binding buffer liquid (sky, Beijing is that Time Inc. reclaims test kit), 65 ℃ of water-baths are dissolved gel fully and are kept pH value of solution between 5.0～6.0;

(5) sol solutions is moved into separator tube, centrifugal 1 minute of 12000g discards the liquid in the collecting pipe;

(6) add 500 μ l lavation buffer solutions (sky, Beijing is that Time Inc. reclaims test kit), centrifugal 1 minute of 12000g discards the liquid in the collecting pipe;

(7) repeating step (6);

(8) 12000g is centrifugal 1 minute, and another clean 1.5ml EP of separator tube dislocation manages, and adds the TE buffer of 20 μ l, hatches 10 minutes for 65 ℃, and centrifugal 1 minute of 12000g gets 2 μ l electrophoresis, and the UVP UV scanning is checked recovering effect.

4.PCR the clone of product

1) coupled reaction

Reclaiming production concentration according to PCR is respectively 1: 2～10 principle by external source fragment and carrier mole ratio with the pMD18-T carrier, design coupled reaction system is as follows: (PCR test kit and pMD18-T support agent box are pressed the operation of test kit operation instructions all available from Dalian TaKaRa company)

The purpose fragment connects:

Reclaim fragment (200ng/ μ l 1 μ l

pMD18-T(50ng/μl) 1μl

ddH ₂O 3μl

Connect solution (the Dalian TaKaRa pMD18-T of company support agent box) 5 μ l

Cumulative volume 10 μ l

The connection of section (Control insert) DNA is inserted in contrast:

Contrast insert DNA (50ng/ μ l 1 μ l

pMD18-T(50ng/μl) 1μl

ddH ₂O 3μl

Connect solution (the Dalian TaKaRa pMD18-T of company support agent box) 5 μ l

Cumulative volume 10 μ l

16 ℃ connect 3 hours.

2) screening, the evaluation of conversion of connection product and recon

(1) preparation (CaCl of bacillus coli DH 5 alpha competence bacteria ₂Method)

The aseptic inoculation ring dips in and gets-70 ℃ of frozen DH5 α and protects kind of a liquid, and the trilinear method streak inoculation was cultivated 12～16 hours for 37 ℃ in the LB flat board.

The single colony inoculation of picking is in 2ml LB culture fluid, and 37 ℃ of shaking tables were cultivated 12～16 hours.

With the DH5 α of incubated overnight in 1% ratio transferred species to the LB culture fluid, 37 ℃ of shaking tables are cultured to OD ₆₀₀Be 0.2～0.4 o'clock, 800g collected antibacterial in centrifugal 5 minutes.

The 0.1M CaCl that adds the 1ml pre-cooling ₂Resuspended precipitation, ice-water bath 3 hours.Centrifugal 5 minutes of 4 ℃ of 800g abandon supernatant.The 0.1M CaCl that adds 100 μ l pre-coolings ₂Suspend and precipitate, ice-water bath 1 hour, standby.

(2) contain the Amp of X-Gal, IPTG ⁺The preparation of LB flat board

The preceding fusing of LB solid medium treats that temperature reduces to about 50 ℃ that to add Amp be 100mg/L to final concentration, and the mixing hypsokinesis is to dull and stereotyped, natural coagulation.Use and got Amp in preceding 2～3 hours ⁺The LB flat board adds 40 μ l X-Gal (20mg/ml), 5 μ lIPTG (200mg/ml), with the coating of L rod evenly, places 37 ℃ of incubators standby.

(3) connecting product transforms

Get each 100 μ l of tee pipe competence bacteria liquid simultaneously respectively, first pipe adds the coupled reaction product, and the second pipe adding contrast is inserted section (control insert) DNA and connected product, as positive control, tee pipe does not add foreign DNA, as negative control, and ice-water bath 60 minutes.42 ℃ of water-bath heat shocks 90 seconds were placed ice-water bath rapidly 1～2 minute.Every pipe adds 700 μ l LB culture fluid, and 37 ℃ of shaking tables were cultivated 1 hour.Mixing precipitated after each pipe centrifugal 1 minute with 800g, suction were abandoned 400 μ l supernatants, respectively got 50 μ l coating Amp ⁺The LB flat board, 37 ℃ of incubator overnight incubation.

(4) screening of purpose recon and evaluation

1. picking connection product transforms dull and stereotyped good indigo plant, the white macula bacterium colony of separation of going up and inoculates Amp respectively ⁺The LB flat board was cultivated 12～16 hours for 37 ℃.Transferred species is in Amp ⁺In the LB culture fluid, 37 ℃ of shaking table overnight incubation.

2. plasmid DNA extracting (using Omega company plasmid extraction test kit) is the blue white macula plasmid of extracting respectively

Get bacterium liquid and be sub-packed in the 1.5ml centrifuge tube, centrifugal 3 minutes of 12000g leaves and takes precipitation.

Every pipe adds 100 μ l solution I (Omega company plasmid extraction test kit) and suspends, and mixing fully vibrates.

Add 100 μ l solution II (Omega company plasmid extraction test kit), soft mixing, ice-water bath 5 minutes.

Add 250 μ l solution III (Omega company plasmid extraction test kit), the mixing that gently shakes, room temperature was placed 10 minutes.

Centrifugal 10 minutes of 4 ℃, 12000g move to supernatant in the separator tube.

Centrifugal 1 minute of 12000g topples over the waste liquid in the collecting pipe.

Add 500 μ l lavation buffer solutions (Omega company plasmid extraction test kit) in separator tube, the same centrifugal and discard waste liquid in the collecting pipe.

Repeating step 7.

Centrifugal 1 minute of 12000g volatilizees ethanol fully.

Separator tube is placed another clean EP pipe and add a certain amount of TE buffer (Omega company plasmid extraction test kit), 65 ℃ of water-baths 5 minutes, centrifugal 1 minute of 12000g.

Get a certain amount of eluent and carry out electrophoresis, all the other place-20 ℃ of preservations standby.

3. enzyme action is identified: respectively locus coeruleus plasmid DNA and white macula plasmid DNA are carried out double digestion.(restricted enzyme is available from Dalian TaKaRa company)

Locus coeruleus plasmid DNA 1 μ l

BamHI 1μl

10 * buffer (K), 1 μ l

ddH ₂O 7μl

Cumulative volume 10 μ l

Recombinant plasmid dna 5 μ l

NdeI or NcoI 0.5 μ l

XhoI 0.5μl

10 * buffer (K), 1 μ l

ddH ₂O 3μl

Cumulative volume 10 μ l

Behind the mixing, 37 ℃ of water-baths 4 hours.

5.PCR the sequence analysis of product

The positive bacterial strain that transforms of TA clone is delivered to company, extract plasmid according to a conventional method, adopt the terminal cessation method of two deoxidations, carry out sequencing inserting fragment.The pcr amplification effect is shown in accompanying drawing 2,3,4,7,11.

The acquisition of embodiment 2 intramolecularly adjuvant fusion gene AhpC-LTB (AL)

Design of primers is with synthetic

AhpC P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

P2’ 5’- GCTACCTCCGCCTCCAAGCTTAATGGAAT

Linker

LTB P5” 5’- GGAGGCGGAGGTAGCGCTCCCCAGTCTATT-3’

linker

P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

AhpC and the LTB that reclaims with embodiment 1 is template respectively, with P1 and P2 ', P5 " and P6 primer increase respectively AhpC and LTB gene, the bacterial genomes extracting is that epoch centrifugal column type bacterial genomes DNA extraction test kit carries out by the sky.The pcr amplification system is: template DNA 1 μ l; 10 * PCR buffer, 5 μ l; DNTPs (10mmol/L) 4 μ l; Each 1 μ l of primer (0.025mmol/L); Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l; Add deionized water to final volume 50 μ l.

With the reaction system mixing, after the centrifugal treating, add 30 μ l paraffin oil.94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 40 seconds, 72 ℃ were extended 40 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Get 1 μ l product after reaction finishes, 1.5% agarose gel electrophoresis detects the PCR effect.

AhpC that has Linker and LTB gene with recovery are template, and P1 and P6 are that primer carries out overlap extension PCR reaction.The pcr amplification system is the same.

The overlap extension pcr amplification reaction: 94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 70 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Behind the agarose gel electrophoresis, reclaim the purpose fragment.

The clone and the sequence analysis of PCR product are the same.Overlap extension PCR the results are shown in shown in the accompanying drawing 8.

The acquisition of embodiment 3 intramolecularly adjuvant fusion gene AhpC-CTB (AC)

Design of primers is with synthetic

AhpC P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

P2’ 5’- GCTACCTCCGCCTCCAAGCTTAATGGAAT

Linker

CTB P7” 5’- GGAGGCGGAGGTAGCATTAAATTAAAATTTGGT-3’

linker

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

AhpC and the CTB that reclaims with embodiment 1 is template respectively, with P1 and P2 ', P7 " and P8 be primer, increase respectively AhpC and CTB gene, the bacterial genomes extracting is that epoch centrifugal column type bacterial genomes DNA extraction test kit carries out by the sky.The pcr amplification system is: template DNA 1 μ l; 10 * PCR buffer (containing magnesium chloride), 5 μ l; DNTPs (10mmol/L) 4 μ l; Each 1 μ l of primer (0.025mmol/L); Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l adds deionized water to final volume 50 μ l.

With the reaction system mixing, after the centrifugal treating, add 30 μ l paraffin oil.94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 40 seconds, 72 ℃ were extended 40 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Get 1 μ l product after reaction finishes, 1.5% agarose gel electrophoresis detects the PCR effect.

AhpC that has Linker and CTB gene with recovery are template, and P1 and P8 are that primer carries out overlap extension PCR reaction.The pcr amplification system is the same.

The overlap extension pcr amplification reaction: 94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 70 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Behind the agarose gel electrophoresis, reclaim the purpose fragment.The clone and the sequence analysis of PCR product are the same.

The acquisition of embodiment 4 fusion gene AhpC-NapA (AN)

Design of primers is with synthetic

AhpC P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

P2’ 5’- GCTACCTCCGCCTCCAAGCTTAATGGAAT

Linker

NapA P3’ 5’-T GGAGGCGGAGGTAGCAAAACATTTGAAATT-3

linker

P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

The AhpC and the NapA gene that reclaim with embodiment 1 are template, are that primer carries out overlap extension PCR and reacts with P1 and P2 ', P3 ' and P4 respectively.The pcr amplification system is: template DNA 1 μ l; 10 * PCR buffer, 5 μ l; DNTPs (10mmol/L) 4 μ l; Each 1 μ l of primer (0.025mmol/L); Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l adds deionized water to final volume 50 μ l.

With the reaction system mixing, after the centrifugal treating, add 30 μ l paraffin oil.94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 40 seconds, 72 ℃ were extended 40 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Get 1 μ l product after reaction finishes, 1.0% agarose gel electrophoresis detects the PCR effect.

AhpC that has Linker and NapA gene with recovery are template, and P1 and P4 are that primer carries out overlap extension PCR reaction.The pcr amplification system is the same.

The overlap extension pcr amplification reaction: 94 ℃ of pre-degeneration 5 minutes, 30 seconds s of 94 ℃ of degeneration, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 70 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Behind the agarose gel electrophoresis, reclaim the purpose fragment.

The clone and the sequence analysis of PCR product are the same.

The acquisition of embodiment 5 intramolecularly adjuvant fusion gene AhpC-NapA-LTB (AUL)

Design of primers is with synthetic

AhpC-NapA P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

P4’ 5’- GCTACCTCCTCCACTTCCGCCTCCAGCCAAATGGGCTTCTAG-3’

linker

LTB P5’ 5’- GGAGGCGGAAGTGGAGGAGGTAGCGCTCCCCAGTCTATT-3’

linker

P6 5’- CTCGAGGTTTTCCATGCTGATTCC-3’

XhoI

The LTB genome that AhpC-NapA gene that reclaims with embodiment 4 and embodiment 1 reclaim is a template, is primer amplification AhpC-NapA and LTB gene with P1 and P4 ', P5 ' and P6.The pcr amplification system is: template DNA 1 μ l; 10 * PCR buffer, 5 μ l; DNTPs (10mmol/L) 4 μ l; Each 1 μ l of primer (0.025mmol/L); Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l adds deionized water to final volume 50 μ l.

With the reaction system mixing, after the centrifugal treating, add 30 μ l paraffin oil.94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 40 seconds, 72 ℃ were extended 70 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Get 1 μ l product after reaction finishes, 1.0% agarose gel electrophoresis detects the PCR effect.

AhpC-NapA fusion gene that has joint and LTB with recovery are template, and P1 and P6 are that primer carries out overlap extension PCR reaction.The pcr amplification system is the same.

The overlap extension pcr amplification reaction: 94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 90 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Behind the agarose gel electrophoresis, reclaim the purpose fragment.

The clone and the sequence analysis of PCR product are the same.Overlap extension the results are shown in shown in the accompanying drawing 9,10.

The acquisition of embodiment 6 intramolecularly adjuvant fusion gene AhpC-UreB414-CTB (AUC)

Design of primers is with synthetic

AhpC-NapA P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

P4’ 5’- GCTACCTCCTCCACTTCCGCCTCCAGCCAAATGGGCTTCTAG-3’

linker

CTB P7’ 5’- GGAGGCGGAAGTGGAGGAGGTAGCATTAAATTAAAATTTGGT-3’

linker

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

The CTB genome that AhpC-NapA gene that reclaims with embodiment 4 and embodiment 1 reclaim is a template, is primer amplification AhpC-NapA and CTB gene with P1 and P4 ', P7 ' and P8.The pcr amplification system is:

Template DNA 1 μ l; 10 * PCR buffer, 5 μ l; DNTPs (10mmol/L) 4 μ l; Each 1 μ l of primer (0.025mmol/L); Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l adds deionized water to final volume 50 μ l.

With the reaction system mixing, after the centrifugal treating, add 30 μ l paraffin oil.94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 40 seconds, 72 ℃ were extended 70 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Get 1 μ l product after reaction finishes, 1.5% agarose gel electrophoresis detects the PCR effect.

AhpC-NapA fusion gene that has Linker and CTB with recovery are template, and P1 and P8 are that primer carries out overlap extension PCR reaction.The pcr amplification system is the same.

The overlap extension pcr amplification reaction: 94 ℃ of pre-degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 90 seconds, 35 circulations, 72 ℃ were extended 10 minutes fully.Behind the agarose gel electrophoresis, reclaim the purpose fragment.

The clone and the sequence analysis of PCR product are the same.

The structure and the screening of embodiment 7 recombinant gene expression plasmids and efficient expression engineering

1. construction of recombinant plasmid

To contain the pMD-18T carrier of genes of interest and expression vector pET-28a (+) or pET-22b (+) (available from U.S. Novagen company, preserve this chamber) double digestion, the enzyme action product is after 1.0% sepharose electrophoresis, purpose fragment glue reclaim purification, connect with ligase, transformed into escherichia coli DH5 α, extract plasmid, double digestion, 1.0% agarose gel electrophoresis is identified.

Relevant operation concrete steps are as follows:

1) plasmid DNA extracting (use Omega company plasmid extraction test kit, press the operation of test kit operation instructions)

(1) separates good bacterium colony transferred species on the picking flat board in being with corresponding antibiotic LB culture fluid, 37 ℃ of shaking table overnight incubation;

(2) get bacterium liquid and be sub-packed in the 1.5mL centrifuge tube, centrifugal 3 minutes of 12000g leaves and takes precipitation;

(3) every pipe adds 100 μ L solution I (Omega company plasmid extraction test kit) suspension, and mixing fully vibrates;

(4) add 100 μ L solution II (Omega company plasmid extraction test kit), soft mixing, ice-water bath 5 minutes;

(5) add 250 μ L solution III (Omega company plasmid extraction test kit), the mixing that gently shakes, room temperature was placed 10 minutes;

Centrifugal 10 minutes of (6) 4 ℃, 12000g move to supernatant in the separator tube;

(7) 12000g is centrifugal 1 minute, topples over the waste liquid in the collecting pipe;

(8) add 500 μ L lavation buffer solutions (Omega company plasmid extraction test kit) in separator tube, the same centrifugal and discard waste liquid in the collecting pipe, repeated washing once;

(9) 12000g is centrifugal 1 minute, and ethanol is volatilized fully;

(10) separator tube is placed another clean EP pipe and add a certain amount of TE buffer (Omega company plasmid extraction test kit), 65 ℃ of water-baths 5 minutes, centrifugal 1 minute of 12000g;

(11) get a certain amount of eluent and carry out electrophoresis, all the other place-20 ℃ of preservations standby.

2) agarose gel electrophoresis:

1.0% agarose gel, 1 * TAE buffer, 120-150mA, electrophoresis 20-40 minute.

50 * TAE storage liquid prescription: 2.0mol/L Tris alkali, 1.0mol/L NaAc, 0.1mol/L Na ₂EDTA; Regulate pH8.3 with glacial acetic acid.

3) endonuclease reaction of plasmid DNA:

1 μ g plasmid DNA

1 μ l, 10 * buffer (TaKaRa restriction endonuclease reagent)

1 μ l restricted enzyme Nco I/XhoI or NdeI/XhoI (10u/ μ l)

Mend to 10 μ l with distilled water

Mixed back 37 ℃ of incubation 1-2 hours.

4) target DNA of sepharose electrophoresis glue reclaims purification:

Under uviol lamp, observe and downcut the target DNA electrophoresis band on the agarose gel, move into 1.5mL EP pipe.

Add Omega company glue and reclaim the DNA binding buffer liquid of test kit, 65 ℃ of water-baths are dissolved gel fully and are kept pH value of solution between 5.0～6.0.Sol solutions is moved into separator tube, and centrifugal 1 minute of 12000g discards the liquid in the collecting pipe.

Add supporting lavation buffer solution (Omega company plasmid extraction test kit), centrifugal 1 minute of 12000g discards the liquid in the collecting pipe.Repeated washing 1 time.

Centrifugal 1 minute of 12000g, another clean 1.5mL EP pipe of separator tube dislocation, the TE buffer of adding certain volume was hatched 10 minutes for 65 ℃, and centrifugal 1 minute of 12000g gets a certain amount of electrophoresis, and the UVP ultraviolet scanner detects and reclaims purification effect.

5) coupled reaction (use Shanghai to give birth to worker company and connect test kit)

By the concentration of UV spectrophotometer measuring target DNA fragment and carrier segments, be generally 1: 2～10 principle according to external source fragment and carrier mole ratio, design coupled reaction system is as follows:

Target DNA 1 μ l; Plasmid vector 1～2 μ l; Connect solution 5 μ l; DdH ₂O 2～3 μ l, cumulative volume 10 μ l.22 ℃ connect 12-16 hour.

6) preparation (CaCl of competence bacteria ₂Method)

(1) the aseptic inoculation ring dips in and gets-70 ℃ of frozen antibacterials guarantor kind of liquid, and the trilinear method streak inoculation is cultivated 12～16h for 37 ℃ in the LB flat board.

(2) the single colony inoculation of picking is in 2mL LB culture fluid, and 37 ℃ of shaking tables are cultivated 12～16h.

(3) with the DH5a of incubated overnight in 1% ratio transferred species to the LB culture fluid, 37 ℃ of shaking tables are cultured to OD ₆₀₀To 0.2～0.4 o'clock, 8000g collected antibacterial in centrifugal 5 minutes.

(4) the 0.1mol/L CaCl of adding 1ml pre-cooling ₂Resuspended precipitation, ice-water bath 3 hours.Centrifugal 5 minutes of 4 ℃ of 8000g abandon supernatant.The 0.1mol/L CaCl that adds 100 μ l pre-coolings ₂Suspend and precipitate, ice-water bath 1 hour, standby.

7) connecting product transforms

(1) gets competence bacteria liquid 100 μ l, add the coupled reaction product; Ice-water bath 60 minutes, 42 ℃ of water-bath heat shock 100s placed ice-water bath rapidly 1～2 minute.

(2) add 100 μ l LB culture fluid, 37 ℃ of shaking tables were cultivated 1 hour.

(3) with 8000g centrifugal 10 minutes, inhale and abandon mixing precipitation behind the 100 μ l supernatants, respectively get 50 μ l spread plates, 37 ℃ of incubator overnight incubation.

2. efficiently express the structure and the screening of fusion rotein engineering bacteria

To contain in recombinant plasmid transformed e. coli bl21 (DE3) competence bacteria of purpose fusion gene, through Amp ⁺Or K ⁺The LB plate screening extracts plasmid enzyme restriction and identifies.

The reorganization bacterium of identifying of learning from else's experience is inoculated in 3ml and contains in the LB culture fluid of Kan or Amp 37 ℃ of shaking table overnight incubation.Contain the recombination engineering of incubated overnight in the LB culture fluid of Kan or Amp in 20ml in 1% ratio transferred species next day, 37 ℃ of shaking tables were cultivated 2.5 hours, induced 5 hours with IPTG, SDS-PAGE detects Expression of Fusion Protein form and expression, the screening efficient expression strain.

Structure and abduction delivering the results are shown in shown in the accompanying drawing 4,5,12,13.

The fermentation of embodiment 8 dna recombinant expression engineering bacterias

Fermentation technology is as follows:

Adopt German B.Braun 10L fermentation tank, plant the inoculation of daughter bacteria 10% ratio in the sweat, keep 45% dissolved oxygen, 37 ℃ of temperature, pH7.4, treat that concentration of glucose reduces at 0.1% o'clock and add IPTG 500 μ mol/L and induce and received bacterium in 4～6 hours.

Sweat on the batch culture basis of cascade dissolved oxygen control, flow feeding.

The used culture medium of sweat is added 0.6% yeast leachate and 2mg/L ZnCl for improvement M9-CAA culture medium on the basis of M9-CAA ₂4H ₂O, 2mg/LCoCl ₂4H ₂O, 4mg/L FeSO ₄16H ₂O, 5mg/L H ₃BO ₃, 1.6mg/LMnCl ₂4H ₂O, 4mg/L CuSO ₄Form.

Bacterium liquid, 4 ℃ centrifugal (8000g) 15 minutes are reclaimed in the fermentation back.Supernatant is abandoned in suction, collects antibacterial, and the back of weighing is frozen standby.

The result: the 10L zymocyte liquid can be gathered in the crops about antibacterial weight in wet base 600 grams.

The purification of embodiment 9 reorganization destination proteins and dosage form preparation

1. cleer and peaceful inclusion body extracts on the solubility: the thalline 200-500g that efficiently expresses is suspended with 1: 10 (W/V) ratio of TE buffer (pH 8.0 for 20mmol/L Tris, 5mmol/L EDTA), adopt the cell homogenates machine to make its mix homogeneously after 4 ℃ of pre-coolings.Adopting high pressure homogenizer is break bacterium (breaking bacterium 4～6 times altogether) under the condition of 70Mpa at pressure, after broken bacterium finishes, bacterium liquid smear staining takes a morsel, the integrity of microscopically observation of cell guarantees that cell breakage is complete, with 500 * g centrifugal 30 minutes subsequently, abandon precipitation, with 15,000 * g centrifugal 40 minutes again, collecting precipitation was inclusion body.Inclusion body uses cleaning mixture A (pH 8.0 for 20mmol/L Tris, 5mmol/LEDTA) and cleaning mixture B (20mmol/L Tris, 2mol/L carbamide, pH 8.0) respectively to wash respectively 2 times with the ratio of 1: 10 (W/V).Wash conditions is: 4 ℃ were stirred 20 minutes, and centrifugal 40 minutes of 15,000 * g collects the inclusion body precipitation; At last inclusion body is used the mixed of solubilization of inclusion bodies liquid (1mmol/L EDTA, 20mmol/L Tris, 8mol/L carbamide pH 8.0) with 1: 10 (W/V), 4 ℃ were stirred 3 hours, and centrifugal 45 minutes of 15,000 * g gets the raw material of supernatant as next step purification.

Inclusion body extracts used buffer:

1) TE buffer: 20mmol/L Tris, 5mmol/L EDTA, pH 8.0

2) inclusion body cleaning mixture A:5mmol/L EDTA, 20mmol/L Tris, 1%Triton X-100, pH 8.0

3) inclusion body cleaning mixture B:20mmol/L Tris, 2mol/L carbamide, pH 8.0

4) solubilization of inclusion bodies liquid: 1mmol/L EDTA, 20mmol/L Tris, 8mol/L carbamide (pH 8.0)

2. metal ion-chelant chromatography: select affinity column Chelating Sepharose to carry out purification, use 20mmol/L Tris, 5mmol/LEDTA, pH7.0～9.0 pair destination protein carries out purification, adopts the imidazoles gradient elution.

3. anion column purification: select anion column HiTrap Q to carry out purification, use 20mmol/L Tris, 5mmol/LEDTA, pH7.0～9.0 pair destination protein carries out purification, adopts the NaCl gradient elution.

4.Superdex gel permeation chromatography desalination: target protein that step 3 obtains in glucosan PEG bag filter, concentrate or ultrafiltration and concentration after filter desalination with solvent resistant column Superdex, take off carbamide and imidazoles.

5. purified target protein is carried out SDS-PAGE, examines and determine its purity.The Lowry method detects protein concentration.

Wherein, the described nickel ion affinity purification of step 2 filler comprises Chelating Sepharose HP, Chelating SepharoseFF;

The described anion purification of step 3 filler comprises Q Sepharose HP, Q Sepharose FF, Q Sepharose XL;

The described gel permeation chromatography post of step 4 comprises Superdex 75, Superdex 200, Superdex HR 10/30.

Purification result as shown in Figure 3.

6. the recombiant protein of said method acquisition is prepared to lyophilization dosage form or capsule respectively.Wherein lyophilization dosage form adds purified water or water for injection dissolving back supplies oral.Lyophilization dosage form preparation method is: add the stabilizing agent mannitol (fructose or sorbitol) of proper proportion (5%～20%) in the destination protein that purification obtained, mixing, aseptic filtration packing postlyophilization; The capsule formulation preparation method is: at first in the destination protein that purification obtained, add the stabilizing agent mannitol of proper proportion (5%～20%), and mixing, aseptic filtration packing postlyophilization refills and inserts enteric coated capsule.

The destination protein expression-form is identified and is seen shown in the accompanying drawing 6,14

Embodiment 10 is added with the preparation of the helicobacter pylori AhpC recombinant vaccine of the outer adjuvant (LTB, CTB) of molecule

1. be the preparation of the helicobacter pylori AhpC recombinant vaccine of adjuvant with LTB: the Hp recombinant antigen protein of purification (rAhpC or rAhpC-NapA) is prepared helicobacter pylori AhpC recombinant vaccine with various combination form and mucosal adjuvants rLTB physical mixed.This vaccine as mucosal adjuvants, is applicable to the immunity inoculation of mucosal route (gastrointestinal tract, nasal mucosa etc.) with rLTB.

2. be the preparation of the helicobacter pylori AhpC recombinant vaccine of adjuvant with CTB: the Hp recombinant antigen protein of purification (rAhpC or rAhpC-NapA) is prepared helicobacter pylori AhpC recombinant vaccine with various combination form and mucosal adjuvants CTB physical mixed.This vaccine as mucosal adjuvants, is applicable to the immunity inoculation of mucosal route (gastrointestinal tract, nasal mucosa etc.) with CTB.

3. the dosage form preparation is with embodiment 9.

Embodiment 11 is added with the preparation of the helicobacter pylori AhpC recombinant vaccine of intramolecularly adjuvant (LTB, CTB)

1. be the preparation of the helicobacter pylori AhpC recombinant vaccine of adjuvant with LTB: the Hp recombinant antigen protein rAhpC-NapA-LTB of purification is as helicobacter pylori AhpC recombinant vaccine.This vaccine is applicable to the immunity inoculation of mucosal route (gastrointestinal tract, nasal mucosa etc.).

2. be the preparation of the helicobacter pylori AhpC recombinant vaccine of adjuvant with CTB: the Hp recombinant antigen protein rAhpC-NapA-CTB of purification is as helicobacter pylori AhpC recombinant vaccine.This vaccine is applicable to the immunity inoculation of mucosal route (gastrointestinal tract, nasal mucosa etc.).

3. the dosage form preparation is with embodiment 9.

The destination protein purification result is seen shown in the accompanying drawing 15

The experiment of embodiment 12 biologic activity

1, specific immune response after the BALB/c mouse immunity

With different dosage form recombinant vaccine oral immunity BALB/c mouse, 150 μ g/ only, respectively at the 0th, 1,2,4 each immunity of week once, 1 week was gathered blood, gastrointestinal tract flushing liquor and feces extract after the last immunity, ELISA detects in the serum specific antibody sIgA level among specific antibody IgG, the IgA and gastrointestinal tract flushing liquor and feces extract.Simultaneously, set up matched group.

Helicobacter pylori AhpC recombinant vaccine test group mice specific antibody IgG, the IgA of various combination and sIgA level are compared with the PBS matched group and are differed extremely significantly (P＜0.001) after the result immunity 4 times, excite mice generation mucosa local immune response and systemic immunne response after pointing out different vaccine oral immunities.Simultaneously from the testing result of different immune indexes think excite after oral mice with the equilibrated antigen-specific immune responses of Th1, Th2.

2, mongolian gerbil oral immunity counteracting toxic substances protection experiment

With different dosage form recombinant vaccine immunity mongolian gerbil, respectively at the 0th, 1,2,4 each immunity of week once, back 10 days of last immunity is with helicobacter pylori adapted strain M13 counteracting toxic substances, cutd open animal extremely in 30 days behind the counteracting toxic substances, get gastric tissue and carry out antibacterial culturing, PCR inspection and check pathological section.The matched group infection rate is 100% as a result, and it is as shown in table 1 that each organizes immune protective effect:

Counteracting toxic substances immune protective effect behind the table 1 oral immunity mongolian gerbil

Sequence number	Group	The number (only) of surviving behind the counteracting toxic substances	Negative (only)	Positive (only)	Protective rate (%)
						1 2 3 4 5	Matched group rAhpC rLTB rCTB rAhpC+rLTB	30 30 29 27 29	0 4 0 0 10	30 26 29 27 19	- 13.3 - - 34.5

6 7 8 9 10 11 12 13 14

rAhpC+rCTB rAhpC-LTB rAhpC-CTB rAhpC+rNapA rAhpC-NapA rAhpC+rNapA+rLTB rAhpC+rNapA+rCTB rAhpC-NapA-LTB rAhpC-NapA-CTB

28 30 29 30 30 29 29 30 30

7 11 8 14 15 21 19 25 23

21 19 21 16 15 8 10 5 7

25.0 36.7 27.6 46.7 50.0 72.4 * 65.6 * 83.3 * 76.7 *

The result shows that the single subunit immune group of rAhpC only obtains 13.3% immune protective rate, and the immune protective rate of AhpC and NapA physical mixed or fusion immune group brings up to 46.7% and 50.0%.As if prove that a kind of antigen immune is difficult in human body and induces enough protective immunities, multiple antigen combination can cause more effectively protective immunity.

After rAhpC mixes with mucosal adjuvants LTB or CTB or merges; immune protective rate is compared with single subunit immune group; protective rate brings up to 34.5%, 25.0% and 36.7%, 27.6%; the immune protective rate of AhpC and NapA and mucosal adjuvants LTB or CTB physical mixed or fusion immune group brings up to 72.4%, 65.6% and 83.3%, 76.7%; above result proves that LTB and CTB have good immunogenicity and potent adjuvant effect, can strengthen immunogenic immunne response ability.Can produce IL-4 but CTB activates Th2, IL-4 then can optionally promote IgE synthetic, thereby thinks that CTB might cause allergy, and LTB does not induce IgE basically, but can start the body fluid and the cellular immunization of body part and whole body effectively.In experiment, relatively, the protective rate of LTB immune group is higher than the CTB immune group, and fusion group protective rate is higher than the physical mixed group between physical mixed group and fusion group for mucosal adjuvants LTB or CTB.Difference is analyzed by statistics between the same antigen various combination mode, differs not remarkable, the immune protective rate height of the more single or two subunit groups of many antigen group, and differ remarkable.

Conclusion: fusion bacterin antigen A UL immunity mongolian gerbil can produce at the full bacterium of Hp than other each groups and attack effective protective effect; simultaneously the vaccine of amalgamation mode have easy, be convenient to advantage such as operation; avoided a plurality of subunits shortcoming of purification respectively, made purifying process more simple and easy to do.Simultaneously, the albumen after the fusion is better brought into play its immune protective effect, and more single subunit and many subunits physical mixed group produce better immune protective effect.

Sequence table

＜110〉Military Medical Univ No.3, P.L.A

＜120〉a kind of people's helicobacter pylori infections prevention and treatment AhpC multivalent subunit vaccine and preparation method thereof

<160>594

<210>1

<211>594

<212>DNA

<213>E.coli BL21(DE3)ahpC

<220>

<221>gene

<222>(1)...(594)

<400>

ATGTTAGTTACAAAACTTGCCCCCGATTTTAAAGCACCTGCCGTTTTAGG 50

AAACAATGAGGTTGATGAACACTTTGAGCTTTCTAAAAATTTAGGCAAAA 100

ATGGTGCGATTCTTTTCTTTTGGCCAAAAGATTTTACTTTTGTATGCCCT 150

ACAGAAATCATTGCGTTTGACAAAAGAGTGAAAGACTTTCACGAAAAAGG 200

CTTTAATGTGATTGGCGTGTCTATTGACAGCGAGCAAGTGCATTTCGCAT 250

GGAAAAACACCCCTGTGGAAAAAGGCGGTATCGGTCAAGTGTCTTTCCCT 300

ATGGTGGCTGATATTACTAAGAGCATTTCCAGAGATTATGATGTGCTGTT 350

TGAAGAAGCGATCGCTTTGAGAGGCTCTTTTTTGATTGACAAAAACATGA 400

AAGTAAGACACGCAGTGGTCAATGATTTGCCATTAGGTAGGAATGCAGAC 450

GAGATGCTTCGCATGGTAGACGCTCTCTTACACTTTGAAGAACATGGTGA 500

AGTGTGCCCAGCAGGCTGGAGAAAAGGCGATAAAGGCATGAAAGCGACTC 550

ACCAAGGCGTTGCAGAGTATCTCAAAGAAAATTCCATTAAGCTT 594

<160>198

<210>1

<211>198

<212>PRT

<213>E.coli BL21(DE3)AhpC

<220>

<221>CDS

<222>(1)...(198)

<400>

MLVTKLAPDFKAPAVLGNNEVDEHFELSKNLGKNGAILFFWPKDFTFVCP 50

TEIIAFDKRVKDFHEKGFNVIGVSIDSEQVHFAWKNTPVEKGGIGQVSFP 100

MVADITKSISRDYDVLFEEAIALRGSFLIDKNMKVRHAVVNDLPLGRNAD 150

EMLRMVDALLHFEEHGEVCPAGWRKGDKGMKATHQGVAEYLKENSIKL. 198

<160>918

<210>1

<211>918

<212>DNA

<213>E.coli BL21(DE3)ahpC-ltB

<220>

<221>gene

<222>(1)...(918)

<400>

ATGTTAGTTACAAAACTTGCCCCCGATTTTAAAGCACCTGCCGTTTTAGG 50

AAACAATGAGGTTGATGAACACTTTGAGCTTTCTAAAAATTTAGGCAAAA 100

ATGGTGCGATTCTTTTCTTTTGGCCAAAAGATTTTACTTTTGTATGCCCT 150

ACAGAAATCATTGCGTTTGACAAAAGAGTGAAAGACTTTCACGAAAAAGG 200

CTTTAATGTGATTGGCGTGTCTATTGACAGCGAGCAAGTGCATTTCGCAT 250

GGAAAAACACCCCTGTGGAAAAAGGCGGTATCGGTCAAGTGTCTTTCCCT 300

ATGGTGGCTGATATTACTAAGAGCATTTCCAGAGATTATGATGTGCTGTT 350

TGAAGAAGCGATCGCTTTGAGAGGCTCTTTTTTGATTGACAAAAACATGA 400

AAGTAAGACACGCAGTGGTCAATGATTTGCCATTAGGTAGGAATGCAGAC 450

GAGATGCTTCGCATGGTAGACGCTCTCTTACACTTTGAAGAACATGGTGA 500

AGTGTGCCCAGCAGGCTGGAGAAAAGGCGATAAAGGCATGAAAGCGACTC 550

ACCAAGGCGTTGCAGAGTATCTCAAAGAAAATTCCATTAAGCTTGGAGGC 600

GGAGGTAGCGCTCCCCAGTCTATTACAGAACTATGTTCGGAATATCGCAA 650

CACACAAATATATACGATAAATGACAAGATACTATCATATACGGAATCGA 700

TGGCAGGTAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGGGCAACA 750

TTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAAAAAAAAGC 800

CATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCA 850

AAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCG 900

GCAATCAGTATGGAAAAC 918

<160>978

<210>1

<211>978

<212>DNA

<213>E.coli BL21(DE3)ahpC-ctB

<220>

<221>gene

<222>(1)...(978)

<400>

ATGTTAGTTACAAAACTTGCCCCCGATTTTAAAGCACCTGCCGTTTTAGG 50

AAACAATGAGGTTGATGAACACTTTGAGCTTTCTAAAAATTTAGGCAAAA 100

ATGGTGCGATTCTTTTCTTTTGGCCAAAAGATTTTACTTTTGTATGCCCT 150

ACAGAAATCATTGCGTTTGACAAAAGAGTGAAAGACTTTCACGAAAAAGG 200

CTTTAATGTGATTGGCGTGTCTATTGACAGCGAGCAAGTGCATTTCGCAT 250

GGAAAAACACCCCTGTGGAAAAAGGCGGTATCGGTCAAGTGTCTTTCCCT 300

ATGGTGGCTGATATTACTAAGAGCATTTCCAGAGATTATGATGTGCTGTT 350

TGAAGAAGCGATCGCTTTGAGAGGCTCTTTTTTGATTGACAAAAACATGA 400

AAGTAAGACACGCAGTGGTCAATGATTTGCCATTAGGTAGGAATGCAGAC 450

GAGATGCTTCGCATGGTAGACGCTCTCTTACACTTTGAAGAACATGGTGA 500

AGTGTGCCCAGCAGGCTGGAGAAAAGGCGATAAAGGCATGAAAGCGACTC 550

ACCAAGGCGTTGCAGAGTATCTCAAAGAAAATTCCATTAAGCTTGGAGGC 600

GGAGGTAGCATTAAATTAAAATTTGGTGTTTTTTTTACAGTTTTACTATC 650

TTCAGCATATGCACATGGAACACCTCAAAATATTACTGATTTGTGTGCAG 700

AATACCACAACACACAAATACATACGCTAAATGATAAGATATTTTCGTAT 750

ACAGAATCTCTAGCTGGAAAAAGAGAGATGGCTATCATTACTTTTAAGAA 800

TGGTGCAACTTTTCAAGTAGAAGTACCAGGTAGTCAACATATAGATTCAC 850

AAAAAAAAGCGATTGAAAGGATGAAGGATACCCTGAGGATTGCATATCTT 900

ACTGAAGCTAAAGTCGAAAAGTTATGTGTATGGAATAATAAAACGCCTCA 950

TGCGATTGCCGCAATTAGTATGGCAAAT 978

<160>1371

<210>1

<211>1371

<212>DNA

<213>E.coli BL21(DE3)ahpC-napA-ltB

<220>

<221>gene

<222>(1)...(1371)

<400>

ATGTTAGTTACAAAACTTGCCCCAGATTTTAAAGCACCTGCCGTTTTAGG 50

AAAGAATGAGGTTGATGAACACTTTGAGCTTTCTAAAAATTTAGGCAAAA 100

ATGGTGCGATTCTTTTCTTTTGGCCAAAAGATTTTACTTTTGTATGCCCT 150

ACAGAAATCATTGCGTTTGACAAAAGAGTGAAAGACTTTCACGAAAAAGG 200

CTTTAATGTGATTGGCGTGTCTATTGACAGCGAGCAAGTGCATTTCGCAT 250

GGAAAAACACCCCTGTGGAAAAAGGCGGTATCGGTCAAGTGTCTTTCCCT 300

ATGGTGGCTGATATTACTAAGAGCATTTCTAGAGATTATGATGTGCTGTT 350

TGAAGAAGCGATCGCTTTGAGAGGCTCTTTTTTGATTGACAAAAACATGA 400

AAGTAAGACACGCAGTGGTCAATGATTTGCCATTAGGTAGGAATGCAGAC 450

GAGATGCTTCGCATGGTAGACGCTCTCTTACACTTTGAAGAACATGGTGA 500

AGTGTGCCCAGCAGGCTGGAGAAAAGGCGATAAAGGCATGAAAGCGACTC 550

ACCAAGGCGTTGCAGAGTATCTTAAAGAAAATTCCATTAAGCTTGGAGGC 600

GGAGGTAGCAAAACATTTGAAATTTTAAAACATTTGCAAGCGGATGCGAT 650

CGTGTTATTTATGAAAGTGCATAACTTCCATTGGAATGTGAAAGGCACCG 700

ATTTTTTCCATGTGCATAAAGCCACTGAAGAAATTTATGAAGAATTTGCG 750

GACATGTTTGATGATCTCGCTGAAAGGATTGTTCAATTAGGACACCACCC 800

ATTAGTCACTTTATCCGAAGCGCTCAAACTCACTCGCGTCAAAGAAGAAA 850

CTAAAACAAGCTTCCACTCTAAAGACATCTTTAAAGAAATTCTAGGCGAT 900

TACAAACACCTAGAAAAAGAATTTAAAGAGCTCTCTAACACCGCTGAAAA 950

AGAAGGCGATAAAGTAACCGTAACTTATGCGGACGATCAATTAGCCAAGT 1000

TGCAAAAATCCATTTGGATGCTAGAAGCCCATTTGGCTGGAGGCGGAAGT 1050

GGAGGAGGTAGTGCACCCCAGTCTATTACAGAACTATGTTCGGAATATCG 1100

CAACACACAAATATATACGATAAATGACAAGATACTATCATATACGGAAT 1150

CGATGGCAGGTAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGCA 1200

ACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAAAAAAA 1250

AGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGA 1300

CCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATT 1350

GCGGCAATCAGTATGGAAAAC 1371

<160>1431

<210>1

<211>1431

<212>DNA

<213>E.coil BL21(DE3)ahpC-napA-ctB

<220>

<221>gene

<222>(1)...(1431)

<400>

ATGTTAGTTACAAAACTTGCCCCAGATTTTAAAGCACCTGCCGTTTTAGG 50

AAACAATGAGGTTGATGAACACTTTGAGCTTTCTAAAAATTTAGGCAAAA 100

ATGGTGCGATTCTTTTCTTTTGGCCAAAAGATTTTACTTTTGTATGCCCT 150

ACAGAAATCATTGCGTTTGACAAAAGAGTGAAAGACTTTCACGAAAAAGG 200

CTTTAATGTGATTGGCGTGTCTATTGACAGCGAGCAAGTGCATTTCGCAT 250

GGAAAAACACCCCTGTGGAAAAAGGCGGTATCGGTCAAGTGTCTTTCCCT 300

ATGGTGGCTGATATTACTAAGAGCATTTCTAGAGATTATGATGTGCTGTT 350

TGAAGAAGCGATCGCTTTGAGAGGCTCTTTTTTGATTGACAAAAACATGA 400

AAGTAAGACACGCAGTGGTCAATGATTTGCCATTAGGTAGGAATGCAGAC 450

GAGATGCTTCGCATGGTAGACGCTCTCTTACACTTTGAAGAACATGGTGA 500

AGTGTGCCCAGCAGGCTGGAGAAAAGGCGATAAAGGCATGAAAGCGACTC 550

ACCAAGGCGTTGCAGAGTATCTTAAAGAAAATTCCATTAAGCTTGGAGGC 600

GGAGGTAGCAAAACATTTGAAATTTTAAAACATTTGCAAGCGGATGCGAT 650

CGTGTTATTTATGAAAGTGCATAACTTCCATTGGAATGTGAAAGGCACCG 700

ATTTTTTCCATGTGCATAAAGCCACTGAAGAAATTTATGAAGAATTTGCG 750

GACATGTTTGATGATCTCGCTGAAAGGATTGTTCAATTAGGACACCACCC 800

ATTAGTCACTTTATCCGAAGCGCTCAAACTCACTCGCGTCAAAGAAGAAA 850

CTAAAACAAGCTTCCACTCTAAAGACATCTTTAAAGAAATTCTAGGCGAT 900

TACAAACACCTAGAAAAAGAATTTAAAGAGCTCTCTAACACCGCTGAAAA 950

AGAAGGCGATAAAGTAACCGTAACTTATGCGGACGATCAATTAGCCAAGT 1000

TGCAAAAATCCATTTGGATGCTAGAAGCCCATTTGGCTGGAGGCGGAAGT 1050

GGAGGAGGTAGTATTAAATTAAAATTTGGTGTTTTTTTTACAGTTTTACT 1100

ATCTTCAGCATATGCACATGGAACACCTCAAAATATTACTGATTTGTGTG 1150

CAGAATACCACAACACACAAATACATACGCTAAATGATAAGATATTTTCG 1200

TATACAGAATCTCTAGCTGGAAAAAGAGAGATGGCTATCATTACTTTTAA 1250

GAATGGTGCAACTTTTCAAGTAGAAGTACCAGGTAGTCAACATATAGATT 1300

CACAAAAAAAAGCGATTGAAAGGATGAAGGATACCCTGAGGATTGCATAT 1350

CTTACTGAAGCTAAAGTCGAAAAGTTATGTGTATGGAATAATAAAACGCC 1400

TCATGCGATTGCCGCAATTAGTATGGCAAAT 1431

Claims (8)

1, gene engineered poly-valence subunit vaccine of Hp AhpC, the active component that it is characterized in that this vaccine are Heliobacter pylori antigen component alkyl peroxide reductases (AhpC).

2. gene engineered poly-valence subunit vaccine of Hp AhpC according to claim 1, the active component that it is characterized in that this vaccine are the fused proteins that Heliobacter pylori antigen component AhpC and neutrophil activating protein NapA are linked together and obtain by joint sequence.

3. gene engineered poly-valence subunit vaccine of Hp AhpC according to claim 1, the active component that it is characterized in that this vaccine are the intramolecularly adjuvant genetic engineering recombinant fusion proteins that the fusion gene of Heliobacter pylori antigen component AhpC or itself and neutrophil activating protein NapA and adjuvant composition LTB or CTB is merged formation on gene level by joint sequence.

4. adopt the bacterin preparation of helicobacter pylori AhpC gene preparation, it is characterized in that this vaccine by according among the claim 1-3 each the recombinant vaccine active component and one or more bacterin preparations in acceptable diluent and carrier form.

5. prepare the method for the genetic engineering multivalent subunit vaccine among the claim 1-4, mainly may further comprise the steps:

(1) provides pcr amplification, gene clone and the sequence analysis of helicobacter pylori AhpC, NapA protective antigen and E.coli LT B subunit LTB, choleratoxin B subunit CTB genes of interest;

(2) the helicobacter pylori AhpC of step (1) or itself and NapA, the thermo-labile intestinal poison of escherichia coli B subunit LTB, the full gene of choleratoxin B subunit CTB or function fragment separately are connected in prokaryotic expression plasmid or the polygenes fusion expression plasmid;

(3) recombinant expression plasmid that obtains with step (2) transforms suitable host bacteria cell, obtains gene process recombinant bacterial strain;

(4) be suitable for expressing under the condition of desired protein the genetic engineering recombination strain of large scale fermentation incubation step (3);

(5) separate the also recombinant protein of purification step (4) generation, promptly obtain helicobacter pylori AhpC recombinant vaccine.

6. preparation method according to claim 5 is characterized in that in step 1, and the primer that clone AhpC, NapA, LTB, CTB gene use is:

AhpC P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

P2 5’-CG CTCGAGAAGCTTAATGGAAT-3’

XhoI

P2’ 5’- GCTACCTCCGCCTCCAAGCTTAATGGAAT

Linker

NapA P3 5’- CATATGAAAACATTTGAAATT-3’

NdeI

P3’ 5’-T GGAGGCGGAGGTAGCAAAACATTTGAAATT-3

linker

P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

P4’ 5’- GCTACCTCCTCCACTTCCGCCTCCAGCCAAATGGGCTTCTAG-3’

linker

LTB P5 5’- CCATGGCTCCCCAGTCTATTAC-3’

NcoI

P5’ 5’- GGAGGCGGAAGTGGAGGAGGTAGCGCTCCCCAGTCTATT-3’

linker

P5” 5’- GGAGGCGGAGGTAGCGCTCCCCAGTCTATT-3’

linker

P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

CTB P7 5’- CATATGATTAAATTAAAATTTG-3’

NcoI

P7’ 5’- GGAGGCGGAAGTGGAGGAGGTAGCATTAAATTAAAATTTGGT-3’

linker

P7” 5’- GGAGGCGGAGGTAGCATTAAATTAAAATTTGGT-3’

linker

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

7. preparation method according to claim 5 is characterized in that the synthetic primer that gene fusion construct uses in the step 2 is:

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC P2 5’-CG CTCGAGTTAAAGCTTAATGGAAT-3’

XhoI

P3 5’- CATATGAAAACATTTGAAATT-3’

NdeI

NapA P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

P5 5’- CCATGGCTCCCCAGTCTATTAC-3’

NcoI

LTB P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

P7 5’- CATATGATTAAATTAAAATTTG-3’

NcoI

CTB P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-LTB P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

AhpC-CTB Nde I

P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-NapA P4 5’- CTCGAGAGCCAAATGGGCTTCTAG-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-NapA-LTB P6 5’- CTCGAGGTTTTCCATGCTGATTGC-3’

XhoI

P1 5’-CG CATATGTTAGTTACAAAACTTGCC-3’

Nde I

AhpC-NapA-CTB P8 5’- CTCGAGATTTGCCATACTAATTGCG-3’

XhoI

8. preparation method according to claim 5 is characterized in that in the step 4, and described anion purification filler is selected from QSepharose HP, Q Sepharose FF or Q Sepharose XL; Nickel ion affinity purification filler is selected from ChelatingSepharose HP or Chelating Sepharose FF; The gel permeation chromatography post is selected from Superdex 75, Superdex 200 or Superdex HR 10/30.

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