CN100469882C - Preparation and use of conservative region of recombinant helicobacter pylori adhesin - Google Patents
Preparation and use of conservative region of recombinant helicobacter pylori adhesin Download PDFInfo
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- CN100469882C CN100469882C CNB031098924A CN03109892A CN100469882C CN 100469882 C CN100469882 C CN 100469882C CN B031098924 A CNB031098924 A CN B031098924A CN 03109892 A CN03109892 A CN 03109892A CN 100469882 C CN100469882 C CN 100469882C
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Abstract
The present invention is the preparation and use of recombinant pyloric spirobacterium adhesin conservation region, and features that the NDA molecule includes a) nucleotide sequence shown in sequence No. 1, b) nucleotide sequence corresponding to the sequence of a) in the genetic code degeneracy range, or c) nucleotide sequence obtained through cross breeding of sequence of a) and/or b) in strict condition. The present invention has the advantages of cloning pyloric spirobacterium adhesin gene by means of PCR technology, constituting vector for sequence analysis and protein expression analysis, and providing antiserum to prevent pyloric spirobacterium from adhering onto the epithelium cell of stomach mucous membrane.
Description
Technical field
The present invention relates to a kind of preparation and purposes of recombinant helicobacterpylori adhesin conserved regions.
Background technology
Helicobacter pylori (Helicobacter pylori, Hp) infect be chronic gastritis and peptide ulceration main diseases because of, also closely related with the generation of adenocarcinoma of stomach, gastric mucosa dependency lymphoid tissue (MALT) malignant lymphoma.In addition, seroepidemiology studies show that the Hp infection is also relevant with the generation of circulation, breathing, the outer digestive system of gastroduodenal and autoimmune disease.Along with Hp cause of disease status rise, research to its mechanism of causing a disease also becomes deeply, the mechanism of causing a disease of Hp comprises all too many levels, but it is the most key that You Yiqi sticks mechanism, because Hp must at first be colonizated in further its pathogenic effects of performance of people's gastric mucosa, is the prerequisite of Hp field planting in mucosa surface and stick.
Since having been found that gastro-duodenal ulcer is transmissible disease, one of therapeutic purpose are eliminated pathogenic agent with microbiotic exactly.Yet, treat separately with various microbiotic (amoxycilline Trihydrate bp, nitrofuran, furazolidin erythromycin a.o) and to be proved and unsatisfactory, because even only have the 0-15% case can eradicate bacterium in this case.At present, sour blocker (Ompeprazol) and microbiotic (amoxycilline Trihydrate bp) are used in combination the result of treatment that reaches the most successful, this therapy makes clearance rate reach 80% (Malfertheiner, 1994).But eliminating helicobacter pylori with antibiotic therapy is not promising long-term treatment regimen, gives birth to plain patience because bacterium can create antagonism rapidly.
Up to having described several potential helicobacter pylori adhesins, and cloned the neural amino breast of the so-called N-acetyl of encoding sticking-in conjunction with a kind of gene (hpaA) of agglutinin of blood and check order (Evans etc., 1993).It is a kind ofly should be able to discern the protein that contains sialic acceptor on the epithelial cell.
In the triturating of Hp vaccine, find the present gene recombinant antigens urease that adopts, vacuolate cytotoxin, catalase etc., be conceived to block the virulence factor of Hp basically, and estimate less with the closely-related adhesin of Hp field planting.At present the Hp adhesin of bibliographical information is more, and wherein verified has four kinds, comprises the Hp adhesin BabA of unique clear and definite acceptor up to now and sticks and adhesin AlpA, AlpB and HopZ that the inhibition of corresponding antibodies experimental results show that through external; Yet these four kinds of adhesins are not to be present in all bacterial strains, be present in the CagA pathogenicity island male bacterial strain as BabA, and there is variation to a certain degree in they in different bacterial strains.Harry etc. just propose should guard as the antigenic component of Hp vaccine as far back as 1998, promptly are not in the special existence and some bacterial strain.
Summary of the invention
Purpose of the present invention just provides a kind of utilization pcr clone helicobacter pylori adhesin gene, and carrier construction carries out sequential analysis and protein expression analysis to it, improves antiserum(antisera) and can stop helicobacter pylori to adhere to the preparation and the purposes of the gene recombination Hp CB of gastric epithelial cell.
The object of the present invention is achieved like this: dna molecular, it is characterized in that, it comprises: (a) nucleotide sequence shown in the sequence number 1, (b) the corresponding nucleotide sequence of sequence in the scope of genetic code degeneracy and (a), or, (c) under stringent condition and (a) or/and the nucleotide sequence of sequence hybridization (b); (a) a kind of polypeptide that can adhere to people's cell of its coding, (b) carrier contains at least one copy of dna molecular, (c) the cell suppressed by vector transforms: reach (a) nucleotide sequence shown in the sequence number 1, or, (b) with (a) in the nucleotide sequence of sequence generation immunological cross-reaction, (c) polypeptide can adhere to people's cell.
Except nucleotide sequence shown in the sequence number 1 and in genetic code degeneracy scope with the corresponding nucleotide sequence of this sequence, the present invention also is included under the stringent condition dna sequence dna with the hybridization of one of these sequences.Present invention resides under a little wash conditions with the hybridization of one of nucleotide sequence shown in the sequence number 1 or with the nucleotide sequence of corresponding nucleotide sequence hybridization in genetic code degeneracy scope.
Dna molecular of the present invention preferably encode can adhere on people's cell, the polypeptide on people's gastric epithelial cell particularly.In addition, dna molecular of the present invention be preferably on the nucleotide level with nucleotides sequence shown in the sequence number 1 and show at least 70%, preferred at least 90% homology especially.And the length of this dna molecular preferably is at least 45 Nucleotide, preferably at least 50 Nucleotide.
Another purpose of the present invention is the carrier that contains at least one copy of dna molecular of the present invention.This carrier can be to be preferably in expression signal (promotor, operator gene, enhanser etc.) control dna molecular of the present invention position any protokaryon or eukaryotic vector thereon down.The example of prokaryote has and resembles the outer carrier of phage (for example lambda particles phage) such chromosome vector and the karyomit(e) resemble the plasmid, and the cyclic plasmid carrier is particularly preferred.
Carrier of the present invention also can be a for example yeast vector or be suitable for high isocellular carrier (for example, plasmid vector, virus vector, plant vector) of eukaryotic vector.
Another purpose of the present invention is with carrier cell transformed of the present invention.In a preferred embodiment, this cell is prokaryotic cell prokaryocyte, preferred gram-negative prokaryotic cell prokaryocyte, preferred especially Bacillus coli cells.But, on the other hand, cell of the present invention also can be an eukaryotic cell, for example fungal cell's (as yeast), animal or plant cell.
The invention still further relates to polypeptide by dna molecule encode of the present invention.This polypeptide preferably can adhere to people's cell, and comprises aminoacid sequence shown in (a) sequence number 1, or the aminoacid sequence of sequence generation immunological cross-reaction (b) and (a).
Polypeptide of the present invention preferably and nucleotides sequence shown in the sequence number 1 show at least 90% homology.
Polypeptide of the present invention is preferably by following method production: transform a kind of cell with dna molecular of the present invention or carrier, under the condition that polypeptide can be expressed, cultivate cell transformed, from cell or/and isolated polypeptide the culture supernatants.Can obtain the polypeptide of the present invention of fusion polypeptide and non-fusion polypeptide form in this method.
Polypeptide of the present invention also can be used as the original antibody that produces of immunity.Therefore, the invention still further relates to the antibody of anti-polypeptide of the present invention.
The present invention relates to a kind of medicinal compositions on the other hand, and it comprises the dna molecular of the present invention as active substance, polypeptide of the present invention or antibody of the present invention, optionally contains common medical aid matter, thinner, additive and carrier.
On the one hand, medicinal compositions of the present invention can be used for diagnosing helicobacter pylori infection.
On the other hand, this medicinal compositions also can be used for preventing or treating helicobacter pylori infection.Treatment is used, polypeptide or its segment are used for producing initiatively vaccine, or antibody is used to produce passive vaccine.
The present invention has utilization pcr clone helicobacter pylori adhesin gene, and carrier construction carries out sequential analysis and protein expression analysis to it, improves antiserum(antisera) and can stop helicobacter pylori to adhere to gastric epithelial cell and improve immunogenicity and active high and adhesion function advantages of higher.
Design CB special primer obtains the protein sequence of four kinds of (BabA), AlpA, AlpB and HopZ by the DNA recombinant technology, the structure gene of adhesin conserved regions, and it is carried out bioinformatic analysis, further express conserved region gene.Made up the recombinant plasmid of four kinds of common conserved regions of adhesin, order-checking shows CB gene length 588bp; 195 amino acid of this genes encoding all reach more than 50% with the homology of four kinds of adhesin conserved regions, and protein molecular weight is about 22.5KD, has demonstrated good antigenicity and hydrophobicity.Analyzed 836767 sequences, with its homology reach 40% evenly be the Hp sequence.The protein electrophoresis analytical results is found, after inducing, efficiently express relative molecular weight and be 22500 albumen, consistent with expection molecular weight size, the gel autoscan is analyzed, its reorganization adhesin conserved regions albumen accounts for 29.6% of bacterial protein, wherein solubility expression accounts for 21.9% of supernatant, and inclusion body accounts for sedimentary 72.6%.
External and experimentation on animals shows that four kinds of adhesin conserved regions of helicobacter pylori CB is safe, does not have or rarelyr causes the effect of human peripheral T Lymphocyte Apoptosis; Biologically active, can promote helicobacter pylori infection patient peripheral blood lymphocyte propagation, the rising of interleukin-4 level, thereby improve the patient and remove the immunological competence of helicobacter pylori: have immunogenicity, can make mouse produce corresponding antibodies (comprising IgA and IgG), its antiserum(antisera) can stop helicobacter pylori for the sticking of people's gastric epithelial cells, and can be used for prevention and treatment helicobacter pylori infection.The mouse experiment of the immunotherapy effect of the attenuated salmonella typhimurium oral vaccine of expression helicobacter pylori CB shows that it can significantly improve the ratio of mouse spleen lymphocyte CD4/CD8, can detect the special special anti-CB-IgA antibody of a large amount of secretions in the intestinal fluid, its 4 all eradication rate is 53.3%, and nontoxic, side effect.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure of PCR product of the present invention,
Fig. 2 is that two enzymes of heavy resistance plasmid PET-226 of the present invention (+)/CB are identified collection of illustrative plates,
Fig. 3 is the SDS-PAGE analysis chart of CB recombinant protein of the present invention,
Fig. 4 is the nucleotide sequence shown in the sequence 1.
Embodiment
Be described in further detail of the present invention below in conjunction with drawings and Examples: shown in Fig. 1～3, dna molecular, it is characterized in that, it comprises: (a) nucleotide sequence shown in the sequence number 1, (b) the corresponding nucleotide sequence of sequence in the scope of genetic code degeneracy and (a), or, (c) under stringent condition and (a) or/and the nucleotide sequence of sequence hybridization (b); Nucleotides sequence is shown at least 90% homology shown in it and the sequence number 1 on nucleic acid level; Its length is at least 45 Nucleotide; (a) a kind of polypeptide that can adhere to people's cell of its coding, (b) carrier contains at least one copy of dna molecular, and (c) the cell suppressed by vector transforms; Polypeptide, it is coded by dna molecular, it comprises: (a) nucleotide sequence shown in the sequence number 1, or, (b) with (a) in the nucleotide sequence of sequence generation immunological cross-reaction, (c) polypeptide can adhere to people's cell.Produce the method for polypeptide, carrier transforms a kind of cell, cultivates cell transformed under the condition that polypeptide is expressed, and from cell or/and isolate polypeptide the culture supernatants. polypeptide produces the application of antibody and the antibody of polypeptide as immunogen.Medicinal compositions, (a) it comprises dna molecular, polypeptide or the antibody as active substance, optionally comprise common medical aid matter, thinner, additive and carrier, (b) medicinal compositions is used for the application of diagnosing helicobacter pylori infection, and (c) medicinal compositions is used to produce the application of the medicament that prevents or treat that helicobacter pylori infection is used.
Utilize the round pcr amplification common conserved regions of adhesin (called after CB) gene, with its directed pET-22b (+) carrier that inserts, at BL21 (DE3) expression in escherichia coli; Expression product is identified through immunoblotting.
1.1, material
Bacterial strain BL21 (DE3) and plasmid pET-22b (+) (providing by BIO ENGINEERING INST MILITARY) are provided; Helicobacter pylori SS1 is that digestion institute of the entire PLA of Nanfang Hospital of No.1 Military Medical Univ. preserves; Restriction enzyme NotI, NocI and T4DNA polysaccharase, Vent archaeal dna polymerase are available from New England Biolabs company, TaqDNA polysaccharase, dna molecular amount standard λ DNA/EcoR I+Hind III are available from Huamei Bio-Engrg Co.,, agarose, dNTPs, DNA fast purifying test kit are available from Promega company, order-checking plasmid purification test kit is available from German Qiagen company, the AlpA rabbit anti-serum is prepared by Odenbreit S, Hp infected patient serum is from 301 Hospital of PLA, and other reagent is homemade analytical pure.
1.2, the determining of adhesin conserved regions dna sequence dna
Utilize the dna sequence dna of the certified Hp adhesin of ANTHEPROT V4.3c software analysis babA2, find that there is conserved regions in the C-terminal amino acid of the babA2 of adhesin, the C-end structure gene order of choosing AlpA is as template (called after CB).
1.3, the extraction of Hp chromosomal DNA
Scrape on the solid medium and get well-grown Hp bacterium colony, press genomic dna preparation method preparation in a small amount, see reference [1] for details.
1.4, the extraction and the purifying of plasmid
Alkaline denaturation is all adopted in quick extracting and a large amount of preparation of plasmid, sees reference [2] for details.
1.5, the pcr amplification of goal gene
According to conserved region gene sequences Design primer, add suitable restriction enzyme site at its 5 ' end, synthetic by Shanghai Bo Ya company.Sequence is as follows:
Conserved regions 1:5 '-TGG
CCATG GATAAC GCG CTC AAC AAT CAG-3 '
Nco?I
Conserved regions 2:5 '-AG T
GC GGC CGC GAA TGA ATA CCC ATA AGA-3 '
Not?I
The warm start method is carried out PCR, 95 ° of sex change 30s, and 55 ℃ of annealing 30s, 72 ℃ are extended 30s, extend 10min again after 35 circulations.0.8% agarose gel electrophoresis is observed amplification.
1.6, the enzyme of dna fragmentation cuts, connects, the evaluation of conversion and positive colony
See reference [2] for details.Plasmid and goal gene DNA are through Not I and Noc I double digestion, and glass milk reclaims endonuclease bamhi, and the effect of T4 ligase enzyme connects 12h for following 16 ℃, transforms host bacterium BL21 (DE3), and the double digestion evaluation and screening goes out positive colony.
1.7, sequencing and analysis
The recombinant clone plasmid that a large amount of extractings of alkaline lysis are identified through double digestion carries out sequencing with automatic sequencer.
1.8, abduction delivering and SDS-polyacrylamide gel electrophoresis
The CB positive colony is behind the IPTG abduction delivering, and reference literature [1] carries out the SDS-polyacrylamide gel electrophoresis.
1.9, immunoblotting assay
Reference literature [1]
2.1, the amplification of conserved region gene
PCR electrophoretic analysis as a result finds that a band is arranged about 588bp, size fulfills the expectation, and sees Fig. 1.
2.2, construction of recombinant plasmid and enzyme cut evaluation
Behind Not I and Noc I double digestion, the directed insertion in pET-22b (+) carrier of same double digestion obtains recombinant plasmid called after pET-22b (+)/AB with the PCR product.Recombinant plasmid the results are shown in Figure 2 through Not I and Noc I double digestion rear electrophoresis preliminary evaluation.
2.3, the conserved region gene fragments sequence analyzes
Be that template checks order directly with recombinant plasmid pET-22b (+)/AB, obtained the dna sequence dna of cloned sequence, through ANTHEPROT V4.3c software analysis, consistent with the C-end structure gene order of alpA, be respectively 53%, 75% and 59% with the homology of other three kinds of adhesin babA, alpB and hopZ.
2.4, the expression of cb gene in intestinal bacteria
With CB positive clone strain 37 ℃ of overnight incubation in LB nutrient solution (containing the 100ug/ml penbritin), be forwarded in the LB nutrient solution that contains penbritin by 1% then, continue to be cultured to the D value and be 0.6-0.8, add IPTG to final concentration be 0.1mmol/L, abduction delivering 3h, centrifugal collection thalline, get the osmotic shock liquid of its pericentral siphon and thalline after ultrasonic precipitation and the CB of supernatant and purifying carry out 10%SDS-PAGE electrophoretic analysis (Fig. 3).Found that, after inducing, efficiently express relative molecular weight and be 22500 albumen, consistent with expection molecular weight size, the analysis of gel autoscan, CB accounts for bacterial protein 29.6%, and wherein solubility expression accounts for 21.9% of supernatant, and inclusion body accounts for sedimentary 72.6%.
2.5, the immunoblotting assay of cb expressing protein
AlpA work one with one of anti-Hp adhesin of rabbit is anti-, and at M, bar positive reaction band appears in about 22500 places, and rabbit anteserum does not have before the control group immunity.Anti-as one with Hp positive patient serum, normal human serum also reaction zone occurs at the same position place in contrast; And any band is not seen in the normal human serum contrast.
Make up the plasmid-type carrier with pcr amplification technology clone Hp CB, be inserted in the bare plasmid, and at expression in escherichia coli.
The protein Preparation of vector expression is used for the treatment of immunogenicity, the biological activity purposes of helicobacter pylori related disease.
Reference:
[1]Sambrook?J，Fritsch?EF，Maniatis?T.Molecular?cloning：a?laboratory?manual[M].
New?York：Cold?Spring?Harbor?Laboratory?Press，1989：35.
[2] Ausubel FM, Brent R, Kingston RE, et al. face grain husk, Wang Hailin pool.Fine works molecular biology guide [M].
Beijing: Science Press, 1998:39.
Claims (7)
1, a kind of dna molecular is characterized in that, it is a kind of in the following sequence:
(a) nucleotide sequence shown in the sequence number 1, or
(b) the corresponding nucleotide sequence of sequence in the scope of genetic code degeneracy and (a).
2, a kind of by the described dna molecular encoded polypeptide of claim 1.
3, the polypeptide of claim 2 is characterized in that it is the nucleotide sequence encoded polypeptide shown in the sequence number 1.
4, a kind of method that is used to prepare the described polypeptide of claim 3, it is characterized in that transforming a kind of cell with the described dna molecular carrier of claim 1, under the condition that polypeptide is expressed, cultivate cell transformed, and from cell or/and isolate polypeptide the culture supernatants.
5, the polypeptide of one of claim 2～3 produces the application of antibody as immunogen.
6, the polypeptide of one of claim 2～3 produces the antibody of polypeptide as immunogen.
7, a kind of medicinal compositions is characterized in that:
(a) it comprises as the polypeptide of one of the described dna molecular of the claim 1 of active substance, claim 2～3 or the antibody of claim 6, optionally comprises common medical aid matter, thinner, additive and carrier, and
(b) medicinal compositions is used to produce and prevents or treat the medicament that helicobacter pylori infection is used.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998053082A1 (en) * | 1997-05-21 | 1998-11-26 | Daewoong Pharmaceutical Co., Ltd. | A recombinant microorganism expressing an antigenic protein, adhesin |
| CN1200763A (en) * | 1995-09-22 | 1998-12-02 | 马普科技促进协会 | New adhesion from helicobactor pylori |
| US6054134A (en) * | 1996-07-25 | 2000-04-25 | Hsc Research & Development Limited | Haemophilus adhesin protein |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1200763A (en) * | 1995-09-22 | 1998-12-02 | 马普科技促进协会 | New adhesion from helicobactor pylori |
| US6054134A (en) * | 1996-07-25 | 2000-04-25 | Hsc Research & Development Limited | Haemophilus adhesin protein |
| WO1998053082A1 (en) * | 1997-05-21 | 1998-11-26 | Daewoong Pharmaceutical Co., Ltd. | A recombinant microorganism expressing an antigenic protein, adhesin |
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