CN1840541A

CN1840541A - Helicobacter pylori protein and its preparing method and use

Info

Publication number: CN1840541A
Application number: CN 200510017475
Authority: CN
Inventors: 段广才; 张荣光
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2005-03-31
Filing date: 2005-03-31
Publication date: 2006-10-04

Abstract

The disclosed Helicobacter pylori protein comprises the seq2 of amino acid sequence same as following product or homology not less than 60%. This invention has well primer effect and high express efficiency as the ideal tool on research for this antigen. The research result shows strong immunity effect and fit to vaccine antigen.

Description

A kind of helicobacter pylori protein and preparation method thereof and application

Technical field

The present invention relates to a kind of albumen, specifically relate to a kind of helicobacter pylori protein and its production and application.

Background technology

At present, helicobacter pylori (Helicobacter pylori Hp) is the human body chronic gastritis of generally acknowledging and the pathogenic bacterium of peptide ulceration, with being related closely of diseases such as cancer of the stomach and gastric lymphoma, very high infection rate is arranged in global crowd.At present, mainly adopt the infection of antibiotic therapy helicobacter pylori clinically, though curative effect is preferably arranged in a short time,, makes the practicality of this class scheme limited owing to have that bacterial drug resistance constantly increases, the medical expense height, can not prevent problems such as recurrence and superinfection.Therefore, the anti-system of helicobacter pylori infection can only be based upon on the basis of application of effective vaccine.

In vaccine research, screening efficiently, immunizing antigen is critical link.At present, though the immunocompetence of multiple Heliobacter pylori antigen is identified, do not find the gratifying antigenic component of a kind of immune protective effect so far as yet.At present the main drawback of research has: 1. whole cell antigen component complexity, can cause the gastrointestinal mucosal damage behind the per oral inoculation, and helicobacter pylori to cultivate requirement condition higher; 2. the single antigen component of purifying from tropina, efficient is low, the preparation cost height; 3. known gene recombinant antigens immune protective rate is all undesirable.4. gene recombined vector and host bacterium are not suitable for, and make expression efficiency low, or make the expressing protein purification difficult, or make expression product lose the immunocompetence of former native protein.

In addition, also there is defective in the clinical diagnosis of helicobacter pylori infection.Adopt clinically and get stomach mucous membrane by gastroscope and carry out diagnostic method that helicobacter pylori cultivates and not only have traumaticly, be difficult for being accepted by the patient, and the test specification height, recall rate is low; And existing immune diagnostic reagent is subjected to that diagnostic antigen is immunocompetent to be influenced, and diagnosis efficiency has much room for improvement.

It is reported; considerable gram negative bacterium outer membrane protein has good antigenic activity; these outer membrane proteins are as the main target of antibody and immunocyte attack; can mediate the most direct effective killing action of bacterium, be whether the decision immune response has protectiveness to human body key factor.Helicobacter pylori protein of the present invention is a kind of outer membrane protein, and called after HP-C101.Its encoding gene has the conservative property of height, is expected to become important immunizing antigen, but does not all retrieve the report of relevant HP-C101 antigenic activity and immune protective at present both at home and abroad.

Summary of the invention

Purpose of the present invention at first is to provide a kind of helicobacter pylori protein antigen, secondly, also comprise its preparation method and application,, and provide a kind of diagnostic antigen for the development of helicobacter pylori infection diagnostic reagent for the development of helicobacter pylori vaccine provides a kind of immunizing antigen.

Purpose of the present invention can realize by following measure:

Albumen of the present invention, its aminoacid sequence and following helicobacter pylori protein aminoacid sequence seq 2

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

Identical or include with seq 2 homologys be greater than or equal to 60% the aminoacid sequence fragment.

The dietary protein origin that the present invention relates to is in helicobacter pylori, also can derive from other bacterial classification simultaneously, is greater than or equal to 60% requirement with seq 2 homologys as long as its aminoacid sequence satisfies.

Nucleic acid molecules encoding said proteins of the present invention is compared with the nucleic acid molecule of encode such amino acid sequences seq 2, has to be greater than or equal to 60% homology.

The proteic preparation method of the present invention is that arbitrary sequence has the primer of classifying the described protein gene of amplification more than or equal to the nucleotides sequence of 60% homology as among employing and following nucleotide sequences P1, the P2;

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SalI

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

The proteic preparation method of the present invention adopts prokaryotic expression carrier to make up the recombinant expression vector of described protein gene.The present invention is with above-mentioned dna recombinant expression carrier transformed into escherichia coli bacterial strain, implements the recombinant expressed and purifying preparation of described protein gene.

It is prevention, treatment and the diagnosis that is used for the mankind and other Mammals helicobacter pylori infection that albumen of the present invention is used, and comprises vaccine, medicine or the diagnostic reagent of preparation prevention or treatment helicobacter pylori infection.Proteic application of the present invention; be characterised in that: prepare a kind of composition; said composition comprises immunogenicity significant quantity albumen as claimed in claim 1; or comprise the described albumen that keeps anti-helicobacter pylori to infect the modified forms of protective immunity inducibility, optional pharmaceutically acceptable thinner, vaccine carrier and the immunological adjuvant of comprising.Proteic application of the present invention is characterized in that: foregoing is applied to prevention, treatment and the diagnosis of helicobacter pylori infection.

Proteic application of the present invention is characterized in that: prepare the diagnostic reagent or the diagnostic kit of a kind of mankind and other Mammals helicobacter pylori infection, wherein contain the albumen as claimed in claim 1 that is labeled or is coupled on the solid phase carrier.The proteic application of the present invention, it is characterized in that: described albumen is used for the diagnosis of the mankind and other Mammals helicobacter pylori infection, and it comprises the steps: 1. will contact with the body fluid that extracts in human or other mammalian bodies with the described albumen of solid phase carrier bonded claim 1; 2. detect from above-mentioned body fluid and above-mentioned proteantigen bonded antibody.

Advantage of the present invention is as follows:

1. use PCR primer as herein described, successfully be cloned into the gene fragment of helicobacter pylori protein HP-C101, illustrate that the primer usefulness that designs among the present invention is good.

2. the prokaryotic expression system TBl (pMAL-c2X-HP-C101) of Gou Jianing has higher expression efficiency, and the recombinant protein purification of expression is convenient, and the purity of purified product reaches 90%, illustrates that this expression system is preparation and the ideal tools of studying this proteantigen.

3. by subcutaneous immunization, proved that the dna recombinant expression albumen rHP-C101 of helicobacter pylori protein HP-C101 has good immunocompetence, had the vaccine using value mouse.

4. use the mouse model of helicobacter pylori infection; find significantly to reduce the field planting of helicobacter pylori in the mouse stomach with the rHP-C101 immune mouse; can access higher immune protective rate, immune protective effect is similar to the gene fusion expression albumen (rHU) of helicobacter Pylori urease B and heat shock protein(HSP) A.This result of study shows that rHP-C101 has strong immune protective effect, can be used as vaccine antigen.

Description of drawings

The SDS-PAGE of Fig. 1 TBl (pMAL-c2X-HP-C101) abduction delivering product analyzes

(1: the low molecular weight protein (LMWP) standard; The protein ingredient that 2:TBl (pMAL-c2X-HP-C101) induces 3hr; The protein ingredient that 3:TBl (pMAL-c2X) induces 3hr; The 4:TBl thalline is at the protein ingredient of inductor effect 3hr)

The SDS-PAGE of Fig. 2 recombinant protein rHP-C101 purified product analyzes

(1: the rHP-C101 of purifying; The protein ingredient that 2:TBl (pMAL-c2X-HP-C101) induces 3hr; The protein ingredient that 3:TBl (pMAL-c2X) induces 3hr; The 4:TBl thalline is at the protein ingredient of inductor effect 3hr; 5: the low molecular weight protein (LMWP) standard)

Fig. 3 respectively organizes the interior Hp urease activity of mouse stomach-tissue relatively

(1:rHP-C101+rCTB group; The 2:rHU+rCTB group; The 3:rCTB group; 4:column buffer group)

Embodiment

The present invention does with detailed description below in conjunction with drawings and Examples:

Embodiment 1:

Albumen of the present invention is characterized in that: its aminoacid sequence and following helicobacter pylori protein aminoacid sequence seq 2

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

Albumen of the present invention is characterized in that: nucleic acid molecules encoding said proteins is compared with the nucleic acid molecule of encode such amino acid sequences seq 2, has to be greater than or equal to 60% homology.

The proteic preparation method of the present invention is characterized in that: it is that arbitrary sequence has the primer of classifying the described protein gene of amplification more than or equal to the nucleotides sequence of 60% homology as among employing and following nucleotide sequences P1, the P2;

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SalI

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

Protein preparation method of the present invention is characterized in that: it is to adopt prokaryotic expression carrier to make up the recombinant expression vector of described protein gene.

Protein preparation method of the present invention is characterized in that: it is with above-mentioned dna recombinant expression carrier transformed into escherichia coli bacterial strain, implements the recombinant expressed and purifying preparation of described protein gene.

The proteic application of the present invention is characterized in that: described albumen is prevention, treatment and the diagnosis that is used for the mankind and other Mammals helicobacter pylori infection, comprises vaccine, medicine or the diagnostic reagent of preparation prevention or treatment helicobacter pylori infection.

The proteic application of the present invention; it is characterized in that: prepare a kind of composition; said composition comprises immunogenicity significant quantity albumen as claimed in claim 1; or comprise the described albumen that keeps anti-helicobacter pylori to infect the modified forms of protective immunity inducibility, optional pharmaceutically acceptable thinner, vaccine carrier and the immunological adjuvant of comprising.

The proteic application of the present invention is characterized in that: described composition is applied to prevention, treatment and the diagnosis of helicobacter pylori infection.

The proteic application of the present invention is characterized in that: prepare the diagnostic reagent or the diagnostic kit of a kind of mankind and other Mammals helicobacter pylori infection, wherein contain the albumen as claimed in claim 1 that is labeled or is coupled on the solid phase carrier.

The proteic application of the present invention, it is characterized in that: described albumen is used for the diagnosis of the mankind and other Mammals helicobacter pylori infection, and it comprises the steps: 1. will contact with the body fluid that extracts in human or other mammalian bodies with the described albumen of solid phase carrier bonded claim 1; 2. detect from above-mentioned body fluid and above-mentioned proteantigen bonded antibody.

Embodiment 2

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

Identical or include with the seq2 homology be greater than or equal to 70% the aminoacid sequence fragment.

Albumen of the present invention is characterized in that: nucleic acid molecules encoding said proteins is compared with the nucleic acid molecule of encode such amino acid sequences seq 2, has to be greater than or equal to 70% homology.

The proteic preparation method of the present invention is characterized in that: it is that arbitrary sequence has the primer of classifying the described protein gene of amplification more than or equal to the nucleotides sequence of 70% homology as among employing and following nucleotide sequences P1, the P2;

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SalI

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

Embodiment 3,

Albumen of the present invention is characterized in that: its aminoacid sequence and following helicobacter pylori protein aminoacid sequence seq2

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

Identical or include with the seq2 homology be greater than or equal to 80% the aminoacid sequence fragment.

Albumen of the present invention is characterized in that: nucleic acid molecules encoding said proteins is compared with the nucleic acid molecule of encode such amino acid sequences seq2, has to be greater than or equal to 80% homology.

The proteic preparation method of the present invention is characterized in that: it is that arbitrary sequence has the primer of classifying the described protein gene of amplification more than or equal to the nucleotides sequence of 80% homology as among employing and following nucleotide sequences P1, the P2;

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SalI

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

Embodiment 4,

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

Identical or include with the seq2 homology be greater than or equal to 90% the aminoacid sequence fragment.

Albumen of the present invention is characterized in that: nucleic acid molecules encoding said proteins is compared with the nucleic acid molecule of encode such amino acid sequences seq2, has to be greater than or equal to 90% homology.

The proteic preparation method of the present invention is characterized in that: it is that arbitrary sequence has the primer of classifying the described protein gene of amplification more than or equal to the nucleotides sequence of 90% homology as among employing and following nucleotide sequences P1, the P2;

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SaII

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

Embodiment 5,

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

Identical or include with the seq2 homology equal 100% the aminoacid sequence fragment.

Albumen of the present invention is characterized in that: nucleic acid molecules encoding said proteins is compared with the nucleic acid molecule of encode such amino acid sequences seq2, has 100% homology.

The proteic preparation method of the present invention is characterized in that: it be adopt with following nucleotide sequences P1, P2 in the nucleotides sequence of arbitrary sequence with 100% homology classify the primer of the described protein gene of amplification as;

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SalI

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

Embodiment 6,

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SalI

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

Embodiment 7

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’SalI

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’PstI。

Embodiment 8,

One, technical scheme of the present invention

1. the clone of helicobacter pylori HP-C101 gene and order-checking

Adopting this chamber is template from the chromosomal DNA of the isolating helicobacter pylorus bacteria strain of Zhengzhou gastritis sufferer's stomach MEL-Hp27, use PCR method, obtain the HP-C101 gene fragment, be inserted among the cloning vector pNEB193, use the recombinant plasmid transformed intestinal bacteria, recombinant plasmid is carried out enzyme cut evaluation, check order inserting fragment.

2. the purifying of structure, expression and the expression product of helicobacter pylori HP-C101 gene efficient expression carrier and immunocompetence research

From the recombinant plasmid that makes up previously, downcut HP-C101 with restriction enzyme, use dna ligase, goal gene is connected among the prokaryotic expression carrier pMAL-c2X, and transformed into escherichia coli then obtains making the recombinant expression vector of goal gene and maltose binding protein gene fusion expression.Cut and PCR method evaluation recombinant plasmid with enzyme.Adopt IPTG to carry out abduction delivering.Prepare affinity chromatographic column with polysaccharide resins, the recombinant protein rHP-C101 that purifying is expressed.Using the SDS-PAGE method analyzes expression product and purified product.RHP-C101 and helicobacter pylori O antigen immunity small white mouse with purifying carry out Western blot reaction with immune serum for preparing and recombinant protein.

3. recombinant protein rHP-C101 immunoprotection activation analysis

RHU is the helicobacter pylori hspA of gene recombination and the expression product of ureB fusion gene, from other research of this chamber.Adopting recombinant cholera toxin rCTB is immunological adjuvant.Small white mouse is divided into 4 groups, and the mouse of 2 experimental group is used rHP-C101+rCTB, rHU+rCTB peroral immunity respectively, and these antigens all are dissolved in the identical damping fluid; 2 mice in control group difference per os are irritated and are fed rCTB and damping fluid.1 time weekly, immunity is 4 times altogether.Behind last immunity 7d, each group is all carried out challenge infection with helicobacter pylori, and each every its mouse oral is irritated and fed helicobacter pylori bacterium liquid 200 μ l (5 * 10 ⁸CFU), inoculate altogether 2 times, at interval 8hr.14d puts to death mouse behind challenge infection, gets the mouse stomach and does urease qualitative test and sxemiquantitative test, helicobacter pylori cultivation, smear Gram ' s dyeing microscopic examination and histological examination, detects the influence of immunization to helicobacter pylori field planting in the mouse stomach.

4. statistical analysis

Applied statistics software SPSSl2.0, the relatively employing one-way analysis of variance of measurement data, the relatively employing x of enumeration data ²Check or the definite stochastic method of Fisher are inspection level with α=0.05.

Two, be effective

Enzyme is cut and is identified and the sequencing result demonstration that helicobacter pylori HP-C101 gene is cloned among the pNEB193.The nucleotide sequence length of the HP-C101 gene of MEL-HP27 bacterial strain is 561bp, the long 186aa of amino acid sequence coded.

The Seq1:HP-C101 structural gene sequence

atgattaaaa gaattgcttg tattttaagc ttgagcacga gtttagcgct tgctggcgaa 60

gtgaatgggt tttttatggg tgcgggttat cagcaaggtc gttatggccc ttataacagc 120

aattactctg attggcgcca tggcaatgat ctttatggtt tgaatttcaa attaggtttt 180

gtaggctttg ccaataaatg gtttggggct agggtgtatg gctttttaga ttggtttaac 240

acttcaggga ctgagcacac aaaaaccaat ttgctcacct atggtggcgg tggcgatttg 300

attgtcaatc tcattccttt ggataaattc gctctaggtc ttattggtgg cattcagtta 360

gccggaaaca cttggatgtt cccttatgat gtcaatcaaa cgagattcca gttcctatgg 420

aatttaggcg gaagaatgcg cgttggggat cgcagtgcgt ttgaagcggg cgtgaaattc 480

cctatggtta atcagggcag caaagatgta gggcttatcc gctactattc ttggtatgtg 540

gattatgtct ttactttcta a 561

The aminoacid sequence of Seq 2:HP-C101 structural gene coding:

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF：60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

Correct to the efficient expression vector pMAL-c2X-HP-C101 qualification result that makes up.The molecular weight 70kDa of the recombinant protein rHP-C101 that expresses with expression system TBl (pMAL-c2X-HP-C101), the Recombinant Protein Expression amount accounts for 20% of full bacterium total protein respectively, and the recombinant protein purity behind the purifying all reaches 90%.Western blot result shows that this recombinant protein can be discerned by corresponding mouse immune serum and mouse anti helicobacter pylori O antigen serum.The TB1 (pMAL-c2X-HP-C101) that the result of study explanation makes up has higher expression efficiency, and the recombinant protein of expressing has good immunogenicity and immunoreactivity.

The mouse model of using helicobacter pylori infection carries out the immunoprotection experiment; qualitative and sxemiquantitative result of experiment shows uses the rHP-C101 immunized mice; can significantly reduce the amount of growing of deciding of the interior helicobacter pylori of mouse stomach; can obtain 41.2% (7/17) complete protection ratio, basic identical with the immune protective effect 44.4% (8/18) of rHU.

Three, embodiment

Embodiment A: the preparation of the construction and expression product of helicobacter pylori HP-C101 gene expression engineering bacterium

1. material

1.1 bacterial strain

Helicobacter pylori MEL-Hp27 bacterial strain: separate and guarantor's kind in the one routine chronic superficial gastritis patient's body of Zhengzhou this chamber.

Intestinal bacteria TBl bacterial strain: available from Britain New England Biolabs (NEB) company.

1.2 carrier

Cloning vector pNEB193 and prokaryotic expression carrier pMAL-c2X are all available from Britain New EnglandBiolabs company

1.3 reagent material and reagent preparation

1.3.1 reagent material

Tryptones, yeast extract is available from OXOID company, acrylamide, methylene diacrylamide, TEMED, beta-mercaptoethanol is available from Sigma company, the lower molecular weight standard protein, Proteinase K, the RNA enzyme, 1kb DNA ladder is available from Shanghai Sangon company, the Taq enzyme is available from Shanghai Promega, PstI, SalI, EcoRI, Pyrobest DNA polymerase, dNTP, SDS, the T4 dna ligase, isopropyl-(IPTG), indoles-β-D-galactoside (X-gal) is available from TaKaRa company for 5-bromo-4-chloro-3-, and the DNA purification kit is available from Vitagene company.

1.3.2 the preparation of reagent

Inductor IPTG: get 2g IPTG and be dissolved in the 8ml pure water, be settled to 10ml, with 0.22 μ m membrane filtration degerming, after the packing in 4 ℃ of storages.

Recombinant protein purification reagent:

Cross post damping fluid (Colomn buffer): get 5ml 1M Tris-Cl damping fluid, 0.5ml 0.5M EDTA, NaCl2.93g are dissolved in the 200ml distilled water, adjust pH to 7.4, and autoclaving is in 4 ℃ of storages.With preceding adding beta-mercaptoethanol 0.175ml.Solution contains 20mmol/L Tris-Cl, 200mmol/L NaCl, 1MEDTA, 10mmol/L beta-mercaptoethanol.

Suppress to contain the bacteria culture medium of MBP recombinant protein degraded: get the 0.8g Tryptones, the 0.4g yeast extract powder, 0.4g NaCl is dissolved in the 80ml distilled water, transfers pH value to 7.2 with 10M NaOH, autoclaving, 4 ℃ of storages are with before adding 0.8ml 20% glucose.

The preparation of microbial culture, plasmid extraction and SDS-PAGE agents useful for same is all with reference to molecular cloning experiment guide (J. Sa nurse Brooker work, second edition).

1.4 plant and instrument

STRATOS Tgradient thermograde gene-amplificative instrament (German Biomtre company), ABl-310 sequenator (HIT), gel images analyser Gene Genius (U.S. syugene company), SIM-F124 snowflake ice-making machine and MDF-U4086S very low temperature water tank (SANYO GS company), Millipore desk type high speed refrigerated centrifuge (German Heraeus company), SDC-6 numerical control ultra-low temperature constant temperature groove (the new sesame biotechnology research in Shanghai institute), flooring high speed freezing centrifuge (HITACH company), U-2001 ultraviolet-visible pectrophotometer (Japanese HITACH company), TGL-16G table model high speed centrifuge (Anting Scientific Instrument Factory, Shanghai), GNP-9080 type water isolation type electro-heating standing-temperature cultivator (the accurate experimental installation in Shanghai company limited), DK-8D type electric heating constant temperature tank (the accurate experimental installation in Shanghai company limited), HH-42 type fast constant temperature digital display water tank (Changzhou Guohua Electric Appliance Co., Ltd.), ZD-85 type constant temperature oscillator (Changzhou Guohua Electric Appliance Co., Ltd.), the quick vortex mixer of SK-1 type (Jintan, Jiangsu medical apparatus and instruments factory), the automatic gyroscope of PL-2000C type (sky, Jiangyan City, Jiangsu power medicine equipment company limited), candle cylinder formula anaerobic jar (Shanghai), DYY-III type sepharose vertical electrophoresis groove (Liuyi Instruments Plant, Beijing), DYY-III type sepharose horizontal strip electrophoresis groove (Liuyi Instruments Plant, Beijing).

2. experimental technique

2.1 cultivation of helicobacter pylori and preservation

Adopt the domestic helicobacter pylorus bacteria strain of Bu Shi culture medium culturing MEL-Hp27.Helicobacter pylori is cultivated with the method for preserving is auspicious and sees this research third part.

2.2 the extraction of helicobacter pylori chromosomal DNA

Scrape with transfering loop and to get the helicobacter pylori bacterium colonies that 2～3 rings are cultivated with solid medium, be suspended in 0.5ml in the pure water of autoclaving, add 100 μ l SDS (10%), 100 ℃ of water-bath 5min.Add 3 μ l RNA enzymes (50 μ g/ml), 37 ℃ of water-bath 1hr.Add 3 μ l Proteinase Ks (50 μ g/ml), 42 ℃ of water-bath 1hr.Use the saturated phenol of 600 μ l, phenol chloroform, each extracting of chloroform isoamyl alcohol successively respectively once.The precooling dehydrated alcohol and the 60 μ l 2.5M sodium-acetates that add 2 times of volumes are placed 1hr down in-20 ℃.The centrifugal 10min of 15000rpm.With 70% washing with alcohol precipitation 2 times, place under the room temperature, treat that ethanol volatilizees fully after, precipitation is dissolved in the 100 μ l pure water.

2.3PCR primer design

According to the gene order of the helicobacter pylori J99 of Genbank report, applying biological software designs the PCR primer voluntarily, and primer is synthetic by match Parkson, Beijing biotech firm, and underscore partly is a restriction enzyme site.

P15’-GGG GTCGACATGATTAAAAGAATTGCT-3’(SalI)

P25’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’(PstI)

2.4PCR reaction

2.4.1PCR reaction system

Adopt 50 μ l PCR reaction systems: 10 * Buffer, 5.0 μ l, 4 * dNTP, 4.0 μ l, upstream primer 1.5 μ l, downstream primer 1.5 μ l, template DNA 5.0 μ l, pyrobest polymerase 0.25 μ l, deionization H ₂O 32.75 μ l.

2.4.2PCR reaction parameter

The PCR reaction parameter of amplification helicobacter pylori HP-C101 gene: warm start, 95 ℃ of 4min, * 1; 94 ℃ of 50s, 55 ℃ of 40s, 72 ℃ of 40s, * 30; 72 ℃ of 10min, * 1.

2.4.3PCR the evaluation of reaction product

Sepharose with 1.2% is identified the PCR product.Qualification result: the PCR reaction product of helicobacter pylori HP-C101 gene electrophoretic result in sepharose shows that amplified production length is about 560bp.

2.5 the enzyme of the dna fragmentation of purpose is cut and is reclaimed

2.5.1 the enzyme of helicobacter pylori HP-C101 gene PCR product and plasmid vector is cut

Helicobacter pylori HP-C101 PCR product endonuclease reaction system contains PCR product 22 μ l, 10 * Buffer, 4.0 μ l, PstI 2.0 μ l, SalI 2.0 μ l, the sub-H of degranulation ₂O 10 μ L.

The endonuclease reaction system of cloning vector pNEB193 contains pNEB1934 μ L (0.5 μ g/ μ l), 10 * Buffer, 4.0 μ l, PstI 2.0 μ l, SalI 2.0 μ l, deionization H ₂O28 μ l.

2.5.2 the segmental recovery of purpose

Reclaim test kit (Vitagene) purifying with gel DNA and reclaim PCR product and plasmid vector after enzyme is cut.The process that reclaims is as follows: the DNA after enzyme is cut joins in 1% the sepharose, and electrophoresis 50min cuts the gel of purpose fragment position, weighing gel weight under ultraviolet lamp.According to the test kit explanation, every 1mg gel is converted to the volume of 1 μ l, calculates the volume of gel.The DE-A solution that adds 3 times of gel volumes, behind rifle head mixing, 75 ℃ of heating in water bath 8 minutes are interrupted mixing therebetween 2 times.The DE-B solution that adds 0.5 times of gel volume, fully mixing.Be transferred to DNA then and prepare in the pipe, the centrifugal 1min of 3600g.Abandon filtrate, will prepare pipe and put back in the centrifuge tube, add 500 μ l Wl solution, the centrifugal 30s of 3600g abandons filtrate.To prepare pipe and put back in the centrifuge tube, and add 700 μ l W2 solution, the centrifugal 30s of 3600g abandons filtrate.Wash again 1 time with W2.To prepare pipe and put back in the centrifuge tube, the centrifugal 1min of 12000g abandons filtrate.To prepare pipe and put into 1 1.5ml centrifuge tube, the middle position of adorning the DNA adsorption film of band at the preparation pipe drips 25 μ l elutriants, places 1min under the room temperature, and the centrifugal 1min of 12000g is eluted to target DNA in the centrifuge tube.

2.6 preparation competent cell

Select the single bacterium colony of E.coli TBl from the LB culture medium flat plate, be inoculated in the 5ml LB liquid nutrient medium, the 180rpm shake spends the night.Get 1ml bacterium liquid and add in the 100ml LB liquid nutrient medium, 37 ℃, 250rpm cultivates about 80min, during to logarithmic phase (OD600 ≈ 0.5), bacterium liquid is transferred in the polypropylene tube of two 50ml, and Yu Bingzhong places 20min.In 4 ℃, the centrifugal 10min of 4000g pours out supernatant.Ice the CaCl of the 0.1M of precooling with 10ml ₂Resuspended bacterium places ice 30min.In 4 ℃, the centrifugal 10min of 4000g pours out supernatant.The CaCl that adds 4ml 0.1M in every pipe ₂Solution, resuspended bacterium carries out packing by every pipe 200 μ l bacterium liquid.Behind the glycerol adding, place-70 ℃ to store down.

2.7 carrier is connected with purpose is segmental

Helicobacter pylori HP-C101 gene and carrier ligation system: PCR product 3 μ l, 10 * Buffer, 2 μ l, pNEB 1932 μ l, T4DNA ligase 1 μ l, deionization H ₂O 12 μ l.Under 22 ℃, carry out ligation 8hr.

2.8 transform and screening

To connect product and be divided into two parts of 7 μ l and 13 μ l, be added to respectively in two centrifuge tubes that 200 μ l competent cells are housed, and, put and place 30min on ice, again 42 ℃ of water-bath 2min with rifle head mixing.Rapidly centrifuge tube is transferred in the ice cooling 2min.Add 800 μ l LB substratum in every pipe, under 37 ℃, the 180rpm shake is cultivated 45min.The centrifugal 2min of 8000g abandons supernatant 800 μ l, and is with remaining supernatant that bacterium is resuspended.Bacterium liquid is smoothened scribbling in advance on the LB solid medium flat board 40 μ l X-gal and 4 μ l IPTG, that contain penbritin (100 μ g/ml).In 37 ℃ of incubators, cultivate 14hr.

2.9 the evaluation of recombinant plasmid

2.9.1 extraction plasmid

Select white colony, be inoculated into 5ml and contain in the LB liquid nutrient medium of penbritin (100 μ g/ml), 37 ℃, the 200rpm shake is cultivated 8hr.Get 1ml bacterium liquid, the centrifugal 2min of 10000g abandons supernatant.The plasmid extracting solution I 100 μ l that add precooling, fully mixing adds plasmid extracting solution II 200 μ l again, behind the mixing, puts 2min on ice.Add the plasmid extracting solution III 150 μ l of precooling, mixing is placed 3min on ice, the centrifugal 10min of 12000g.Get supernatant, with isopyknic phenol chloroform and each extracting of chloroform isoamyl alcohol 1 time, the centrifugal 5min of 12000g.Get supernatant liquid, add 2 times of volume dehydrated alcohols, behind the mixing, place 2hr in-20 ℃.The centrifugal 5min of 12000g abandons supernatant, precipitates 1 time with 500 μ l, 70% washing with alcohol.The centrifugal 5min of 12000g abandons supernatant, and it is clean to be placed to the ethanol volatilization under the room temperature, adds 40 μ l deionized waters and 3 μ lRNA enzymes (10mg/ml), 37 ℃ of water-bath 30min.The plasmid that extracts places-20 ℃ to store down.

2.9.2 the enzyme of recombinant plasmid is cut evaluation

The evaluation of pNEB193-HP-C101 recombinant plasmid: the plasmid that extracts is carried out double digestion with PstI and SalI, and enzyme is cut product electrophoresis in 1.2% sepharose, carries out Analysis and Identification with the gel images analyser.Qualification result: it is 2 bands that the recombinant plasmid enzyme is cut the rear electrophoresis collection of illustrative plates, and the empty carrier molecular weight was close after a band and enzyme were cut, and the PCR molecular weight of product of another band and gene amplification is close, and oneself is inserted in the cloning vector to prove gene fragment.

2.10 the order-checking of goal gene in the recombinant plasmid

PNEB193-HP-C101 recombinant plasmid: entrust order-checking portion of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd that clone's helicobacter pylori MEL-HP 27 bacterial strain HP-C101 genes are checked order.

The sequencing result of HP-C101 gene:

2.11 the structure of dna recombinant expression carrier

Recombinant plasmid pNEB193-HP-C101 and expression vector pMAL-c2X all carry out double digestion with SalI and PstI, enzyme is cut product carry out electrophoresis with 1% sepharose, with the Vitagene test kit target DNA fragment are reclaimed.The goal gene, the plasmid pMAL-c2X that enzyme are cut purifying connect 16hr in 4 ℃ of water-baths.Get the E.coli TB1 competence bacterium of 200 μ l ordinary methods preparation, the connection product that adds 10 μ l carries out conversion test.Bacterium after transforming is applied on the LB solid medium that contains ammonia taro penicillin (100 μ g/ml), IPTG and X-gal, hatches about 16hr for 37 ℃.

Select white single bacterium colony, be seeded in the LB liquid nutrient medium that contains penbritin (100 μ g/ml), under 37 ℃, 190rpm shakes bacterium 12hr, extracts recombinant plasmid then.Recombinant plasmid is carried out double digestion and PCR evaluation with PstI and SalI.It is 2 bands that the recombinant plasmid enzyme is cut the rear electrophoresis collection of illustrative plates, and the empty carrier molecular weight after a band and enzyme are cut is close, and the PCR molecular weight of product of another band and gene amplification is close.The proof gene fragment is inserted in the expression vector.

2.12 the abduction delivering of goal gene

The positive colony colony inoculation of identifying is contained to 5ml in the LB liquid nutrient medium of penbritin (100 μ g/ml), and under 37 ℃, 180rpm shakes bacterium and spends the night.Get 800 μ l bacterium liquid and join in the identical LB liquid nutrient medium of 80ml, under 37 ℃, 250rpm shakes bacterium 80min, adds the IPTG of 48 μ l, stops after inducing 3hr.

2.1.3 the purifying of recombinant protein

With inducing the bacterium liquid branch that obtains after finishing to install in the centrifuge tube of two 50ml, under 4 ℃, in refrigerated centrifuge, the centrifugal 20min of 4000rpm.Remove supernatant, add 5ml and cross the post damping fluid, put-20 ℃ of following placements and spend the night.Put in the cold water melt after, have children outside the state plan pulverizing.Adopt the trash ice refrigeration in the excusing from death process, working parameter is: power 600W, pulverize 10s, and 10s intermittently, work total time is 200s.Under 4 ℃,, draw supernatant with the centrifugal 20min of 9000g rotating speed.In the syringe of 20ml, put into the asbestos gauge of 3 layers of silanization, add the polysaccharide resins (amylose resin) of 500 μ l, slowly at the uniform velocity add the post damping fluid excessively of 4ml.Add the bacterium supernatant liquor then, abandon filtered solution.Cross post buffer solution elution recombinant protein with what 5ml was added with maltose, collect filtered solution, in-20 ℃ of storages.

2.1.4 the SDS-PAGE of expression product and purified product analyzes

When analyzing the abduction delivering product, get 1ml bacterium liquid, the centrifugal 1min of 4000rpm, abandon supernatant, in precipitation, add the SDS-PAGE sample buffer of 2 times of concentration of 100 μ l distilled water and 100 μ l, behind the mixing, the centrifugal 5min of 10000rpm before 100 ℃ of water-bath 5min, last sample gets the supernatant application of sample.

When analyzing purified product, get the SDS-PAGE sample buffer that adds isopyknic 2 times of concentration in the sample that 20 μ l collect, in 100 ℃ of water-bath 5min, the centrifugal 5min of 10000g gets the supernatant application of sample.

The concentrated glue of employing 5% and 12% separation gel.Adopt 90mV voltage when electrophoresis begins, when treating that bromjophenol blue is swum to separation gel, voltage changes 140mV into, about 4.5hr of electrophoresis total time.Gel dyes with Coomassie brilliant blue R250.

The result: use the molecular weight 70kDa of the recombinant protein rHP-C101 of expression system TB1 (pMAL-c2X-HP-C101) expression, the Recombinant Protein Expression amount accounts for 20% of full bacterium total protein respectively, and the recombinant protein purity behind the purifying all reaches 90%.

Embodiment B: the immune protective effect of helicobacter pylori dna recombinant expression albumen rHP-C101

1. material

1.1 bacterial strain and laboratory animal

Helicobacter pylori

Provided for 3 ages in week by Henan Province's Experimental Animal Center, 20 of male mouse of kunming are about every body weight 16g～18g.

1.2 reagent material and preparation

1.2.1 reagent material

Tryptones, yeast extract available from OXOID company, the fungistat PXB is available from AMRESC company, and vancomycin is available from GIBCO company, and amphotericin B is available from Sigma company, Trimpex is available from Chinese institute for drug control, and the gramstaining test kit is available from Sichuan Mai Ke Science and Technology Ltd..Calf serum is Tianjin H﹠amp; Y Bioisystech Co., Ltd.RHU is helicobacter pylori heat shock protein gene and the urease gene amalgamation and expression albumen that this chamber makes up in other research.Mucosal adjuvants rCTB is so kind as to give by the Huang Liuyu researcher of Military Medical Science Institute

1.2.2 reagent preparation

The preparation of stationary liquid: neutral buffered formaldehyde solution; 37% formaldehyde 100ml, SODIUM PHOSPHATE, MONOBASIC (NaH ₂PO ₄2H ₂O) 4g, Sodium phosphate dibasic (anhydrous) 6.5g is settled to 1000ml with distilled water.Warthin-Starry cma staining reagent:

(1) acetate buffer: get sodium acetate, anhydrous 8.2g, acetic acid 12.5ml is settled to 1000ml with distilled water, and measuring the pH value should be between 3.6～3.8, and dilution is used as working fluid for 100 times during use.

(2) 1%AgNO ₃: get AgNO ₃0.5g, dissolve among the acetate buffer 50ml.

(3) developing solution: comprise following three kinds of solution:

A liquid: Resorcinol 300mg dissolves in acetate buffer 10ml (3%).

B liquid: gelatin 10g dissolves in (5%) among the acetate buffer 200ml.

C liquid: AgNO ₃2g dissolves in (2%) among the acetate buffer 100ml.

The Giemsa staining fluid: get 6g Giemsa dyestuff adding distil water 300ml, jog makes it dissolving, and room temperature storage is used preceding filtration.

The HE staining reagent:

(1) haematoxylin dyeing liquid: earlier the 2.5g Hematorylin is dissolved in the 25ml dehydrated alcohol, the distilled water that has dissolved 2.5g alum is in advance added in the Hematorylin ethanol liquid, after heating is seethed with excitement solution as early as possible, extinguish burning things which may cause a fire disaster, slowly add red precipitate 1.25g, prevent that solution from spilling, continue to boil 2min, immerse beaker in the cold water immediately, to the dye liquor cooling, add the 20ml Glacial acetic acid, with distilled water cumulative volume is transferred to 500ml, room temperature preservation is used preceding filtration.

(2) Eosin Y staining fluid: the 0.75g Eosin Y is dissolved in the 75ml distilled water, adds 25ml95% ethanol then, add 1～2 in Glacial acetic acid.

(3) Hematorylin color separation liquid: get concentrated hydrochloric acid 1ml,, add to 100ml with 75% ethanol.

Urease reagent: peptone 0.1g, NaCl 0.5g, KH ₂PO4 0.2g, glucose 0.1g, urea 2g, adding distil water be to 100ml, 4 ℃ of preservations.With preceding adding 0.5% phenol red 1.5ml.

1.3 instrument and equipment

Histotome (Shanghai Medical Apparatus and Instruments Factory), GNP-9080 type water isolation type electro-heating standing-temperature cultivator (Shanghai accurate experimental installation company limited), candle cylinder formula anaerobic jar (Shanghai), U-2001 ultraviolet-visible pectrophotometer (Japanese HITACH company).

2 experimental techniques

2.1H.pylori cultivation, evaluation and preservation

2.1.1 solid culture

Adopt the brucella broth substratum, compound method is as follows: get Tryptones 2g, yeast extract powder 0.2g, soy peptone 2g, sodium-chlor 1g, S-WAT 0.02g, agar powder 3g, add 200ml distilled water, adjust pH to 7.2,121 ℃ of autoclaving 20min.Treat that substratum is cooled to 50 ℃～60 ℃, add 16ml anti-freezing sheep blood, add microbiotic by following dosage: 0.1ml polymyxin (2500u/L), 0.5ml amphotericin (2mg/L), 0.5ml vancomycin (10mg/L), 0.2ml TMP (5mg/L) fully behind the mixing, pours in autoclaved glass culture dish.Behind the inoculation helicobacter pylori, culture plate is put in the anaerobic jar, cultivated 3d～4d down for 37 ℃.

2.1.2 liquid culture

What the preparation of liquid nutrient medium was different with solid medium is not add agar powder and sheep blood, and adds the foetal calf serum of 10ml in every 100ml substratum.The preceding substratum soon of high pressure is packed into and is had in the Erlenmeyer flask of two ventpipes, add serum and microbiotic behind the high pressure, in the 200ml substratum, add the bacterium liquid that 200 μ l preserve, in culturing bottle, charge into the mixed gas that contains 80% nitrogen, 15% carbonic acid gas and 5% oxygen.Under 37 ℃, 120rpm, 72hr is cultivated in shaking.

2.1.3 the evaluation of helicobacter pylori

Morphology: first visual inspection colonial morphology, the helicobacter pylori bacterium colony of streak inoculation is transparent needle point sample, the about 1mm of diameter.Make nacterial smear then, drip 1 physiological saline on clean glass slide, scrape the helicobacter pylori bacterium colony of getting solid culture with transfering loop, in physiological saline bacterium is smoothened, room temperature is dried, with spirit lamp flame fixed preparation.Sample is found the bacterium red coloration after Gram ' s dyeing, be Gram ' s dyeing negative bacteria.Thalline is rod-short or sea-gull, and majority has tangible bending.Do not see the spherical thalline that becomes is arranged.From form, bacterial condition is good, can be used for the challenge infection mouse.

Biochemical reaction: adopt the urease reaction test.Scrape with transfering loop and to get a ring helicobacter pylori bacterium colony, join in the homemade urease reagent of 2ml, the reagent color in 5 minutes promptly by the light yellow redness that becomes.

2.1.4 preserve

Scrape from the solid culture flat board and to get bacterium colony, in the centrifuge tube that 200 μ l preserve liquid is housed, bacterium is coated with diffusingly, after preserving the liquid mixing, put in-80 ℃ of refrigerators and preserve.During recovery, promptly hold warm melting after from refrigerator, taking out, put then on ice, be inoculated in the substratum as early as possible.The helicobacter pylori that adopt this chamber preserves liquid and contains 10% sucrose and 50% calf serum.

2.2 animal grouping and immunization protocol

1 group: be experimental group, contain 18 of mouse, per os is irritated and is fed rHP-C101+rCTB 200 μ l/times, contains rHP-C101 40 μ g, rCTB 5 μ g.

2 groups: be experimental group, contain 18 of mouse, irritate simultaneously and feed rHU+rCTB 200 μ l//times, contain rHU 40 μ g, rCTB 5 μ g.

3 groups: be the immunological adjuvant control group, contain 8 of mouse, per os is irritated and is fed rCTB 200 μ l//times, contains rCTB 5 μ g.

4 groups: negative control group, contain 18 of mouse, per os is irritated and was fed post damping fluid 200 μ l//times.Cross the prescription of post damping fluid and see second section.

Before peroral immunity, mouse fasting 12hr prohibits water 4hr.Experimental group and control group are carried out immunization protocol by above-mentioned grouping respectively, and in each immunity in the 0th, 7,14,21 day of testing beginning once, immunity recovered drinking water in back 4 hours.

2.3 helicobacter pylori challenge infection

1w after the last immunity, each group is all carried out challenge infection with helicobacter pylori, and fasting 24hr before the challenge infection prohibits water 4hr, and every its mouse oral is irritated and is fed helicobacter pylori bacterium liquid 200 μ l, contains helicobacter pylori thalline 5 * 10 ⁸CFU, 8hr is total to challenge infection 2 times at interval.Attack back 4hr apart from last, remove fasting.Behind challenge infection 14d, put to death mouse.

2.4 the evaluation of bacteria planting

Cut open the belly immediately behind the disconnected neck execution mouse and get stomach, along big curved side the mouse stomach is cut off, with stroke-physiological saline solution flushing gastric content, along the longitudinal axis stomach is cut to 2 parts, portion is put in 10% formalin fixing for histological examination, and another part stomach-tissue is divided into 3 parts again and is respectively applied for urease test, smear and helicobacter pylori culture identification.

2.4.1 the sxemiquantitative and the qualitative test of urease reaction

Get 3mm * 3mm size holostrome stomach-tissue from stomach hole portion earlier, place the 1.5ml centrifuge tube that 500 μ l urease detection reagent are housed, room temperature left standstill 6 hours, measured reagent in the OD of 550nm value.With the stomach-tissue of 5 mouse of handling without experiment, ask for the baseline value of urease reaction with buying goods wholesale into.Add that with OD value mean 2 times standard deviation is as positive discrimination standard.

2.4.2 smear Gram ' s dyeing

Get stomach hole portion stomach-tissue, mucosal surface is attached on the clean slide, push tissue with tweezers, and make it on slide, to slide, behind the smear, seasoning, spirit lamp flame is fixed, Gram ' s dyeing, oily mirror is observed down, and helicobacter pylori is dyed redness.According to the helicobacter pylori morphological specificity, helicobacter pylori is carried out preliminary evaluation.

2.4.3 helicobacter pylori culture experiment

Get stomach hole portion stomach-tissue, mucosal surface is attached on the solid Bu Shi culture medium flat plate, push tissue, on substratum, smear repeatedly, after inoculation is finished, culture plate is placed anaerobic jar, carry out cultivation and the evaluation of helicobacter pylori by aforesaid method with tweezers.

2.4.4 histological examination

Tissue block with 10% formalin conventional fixing after, become the up dehydration of ethanol of Gradient distribution through 70%～100% concentration, dimethylbenzene is transparent, paraffin embedding, preparation paraffin embedded tissues.Make terrace cut slice with paraffin embedded tissues, the wax band that downcuts is swept in the warm water with writing brush,, pick up the wax band with the slide glass that conventional processing is crossed.60 ℃ of roasting sheet 2hr.Dye with following 3 kinds of dyeing processs.

The Warthin-Starry cma staining: paraffin section is gone into the 4min that dewaxes in the dimethylbenzene by certain direction staining rack of packing into, repeats once.Successively through 100%, 100%, the descending entry of 95%, 90%, 80%, 70% ethanol, in tap water, flowing water flushing 3min, clear to cutting into slices.Going into acetate buffer solution washes once.1%AgNO is put in section ₃In the solution, hatch 30min.Behind the gelatin and the mixing of Resorcinol liquid with preparation in advance, add 2% AgNO ₃Liquid is poured into behind the mixing in the staining jar, and the blending ratio of 5% gelatin, 3% Resorcinol and 2% Silver Nitrate is 15: 1: 2.Observe the changing conditions that tissue color is gone up in section under the room temperature.Tissue removes the liquid that develops the color after becoming pale brown look immediately, and with 55 ℃ of Warm Wash 2 times, acetate buffer solution is washed 1 time.With the up successively dehydration of 70%～100% ethanol, dimethylbenzene is transparent, the neutral gum mounting.On the sample, the nucleus of tissue and helicobacter pylori thalline show black, and histocyte endochylema and mucus light yellow.

The Giemsa dyeing of improvement: paraffin section is gone into the 4min that dewaxes in the dimethylbenzene, repeats once.Successively through 100%, 100%, the descending entry of 95%, 90%, 80%, 70% ethanol, in tap water, flowing water flushing 3min, clear to cutting into slices.Go into the 30min that dyes in the 2%Giemsa staining fluid, tap water flowing water flush away dye liquor, 100% ethanol dehydration, dimethylbenzene is transparent, the neutral gum mounting.The helicobacter pylori thalline shows mazarine.

HE dyeing: tissue slice is gone into the 15min that dyes in the haematoxylin dyeing liquid, flowing water flush away dye liquor through dewaxing and the descending entry of gradient Ethanol Treatment.Adopt 1% hydrochloride alcohol color separation.More than the flowing water flushing 1min, to remove the acidic substance in the section, Yihong dyeing 5min, conventional dehydration, transparent, mounting.Karyon is dyed blueness, endochylema and helicobacter pylori levelling pinken.

3 statistical analysis

Measurement data represents that with mean ± standard deviation the relatively employing one-way analysis of variance between each batch total amount data relatively adopts the LSD method in twos between mean.X is adopted in the enumeration data analysis ²Check or the accurate stochastic method of Fisher.With α=0.05 is inspection level.

Use urease test and the results are shown in Table 1 to what mouse stomach-tissue urease activity detected, what each organized mouse stomach-tissue urease activity relatively sees Fig. 3.After the immunity/attack, adopt 4 kinds of detection methods to detect the positive rate of helicobacter pylori field planting in the mouse stomaches, detection the results are shown in Table 2.The detected result of four kinds of methods is also not quite identical.The detected result that this research is cultivated according to helicobacter pylori is calculated various antigenic immune protective rates.The statistic analysis result of protection ratio otherness shows between group: the immune protective rate of rHP-C101+rCTB group, rHU+rCTB group is higher than damping fluid group and rCTB group, difference has statistical significance (P＜0.05), and difference not statistically significant (P＞0.05) between rHP-C101+rCTB group and the rHU+rCTB group; Preserve difference not statistically significant (P＞0.05) between liquid group and the rCTB group.

The detected result of table 1 mouse stomach-tissue urease activity (x ± s)

Table 4.1 Result of urease assay with mouse gastric tissue( x±s)

Group	OD Value
Group	OD Value	RHP-C10l+rCTB rHU+rCTB rCTB group column buffer	0.513±0.216 0.659±0.3411 1.910±0.630 1.980±0.330

Note：rHP-C101+rCTB Group vs rCTB，P＜0.05；rHP-C101+rCTB Group vs columnbuffer，P＜0.05；rHP-C101+rCTB Group vs rHU+rCTB，P＞0.05；rCTB group vs columnbuffer，P＞0.05.

Table 2 is respectively organized the helicobacter pylori field planting situation of mouse

Table 2 Colonjzation of H.pylori in the mice of different groups

Group	Positive rate				Protection rate
	Positive rate					Urease assay	Smear	Culture	Section
	rHP-C101+rCTB rHU+rCTB column buffer rCTB	9/17 9/18 14/15 8/8	8/17 9/18 12/15 7/8	10/17 10/18 15/15 8/8		Urease assay	Smear	Culture	Section	8/17 9/18 13/15 7/8	7/17 8/18 0/15 0/8

Claims

1. an albumen is characterized in that: its aminoacid sequence and following helicobacter pylori protein aminoacid sequence seq 2

MIKRIACILSLSTSLALAGEVNGFFMGAGYQQGRYGPYNSNYSDWRHGNDLYGLNFKLGF： 60

VGFANKWFGARVYGFLDWFNTSGTEHTKTNLLTYGGGGDLIVNLIPLDKFALGLIGGIQL：120

AGNTWMFPYDVNQTRFQFLWNLGGRMRVGDRSAFEAGVKFPMVNQGSKDVGLIRYYSWYV：180

DYVFTF：186

2, a kind of albumen according to claim 1 is characterized in that: nucleic acid molecules encoding said proteins is compared with the nucleic acid molecule of encode such amino acid sequences seq 2, has to be greater than or equal to 60% homology.

3, the proteic according to claim 1 method of a kind of preparation is characterized in that: it is that arbitrary sequence has the primer of classifying the described protein gene of amplification more than or equal to the nucleotides sequence of 60% homology as among employing and following nucleotide sequences P1, the P2;

P1 5’-GGG GTCGACATGATTAAAAGAATTGCT-3’Sal I

P2 5’-GGG CTGCAGTTAGAAAGTAAAGACATA-3’Pst I。

4, protein preparation method according to claim 3 is characterized in that: it is to adopt prokaryotic expression carrier to make up the recombinant expression vector of described protein gene.

5, according to claim 3 or 4 described protein preparation methods, it is characterized in that: it is with above-mentioned dna recombinant expression carrier transformed into escherichia coli bacterial strain, implements the recombinant expressed and purifying preparation of described protein gene.

6, a kind of proteic according to claim 1 application, it is characterized in that: described albumen is prevention, treatment and the diagnosis that is used for the mankind and other Mammals helicobacter pylori infection, comprises vaccine, medicine or the diagnostic reagent of preparation prevention or treatment helicobacter pylori infection.

7, ask 6 described proteic application according to right; it is characterized in that: prepare a kind of composition; said composition comprises immunogenicity significant quantity albumen as claimed in claim 1; or comprise the described albumen that keeps anti-helicobacter pylori to infect the modified forms of protective immunity inducibility, optional pharmaceutically acceptable thinner, vaccine carrier and the immunological adjuvant of comprising.

8, ask 7 described proteic application according to right, it is characterized in that: described composition is applied to prevention, treatment and the diagnosis of helicobacter pylori infection.

9, ask 6 described proteic application according to right, it is characterized in that: prepare the diagnostic reagent or the diagnostic kit of a kind of mankind and other Mammals helicobacter pylori infection, wherein contain the albumen as claimed in claim 1 that is labeled or is coupled on the solid phase carrier.

10, ask 6 described proteic application according to right, it is characterized in that: described albumen is used for the diagnosis of the mankind and other Mammals helicobacter pylori infection, and it comprises the steps: 1. will contact with the body fluid that extracts in human or other mammalian bodies with the described albumen of solid phase carrier bonded claim 1; 2. detect from above-mentioned body fluid and above-mentioned proteantigen bonded antibody.