CN100378223C - Pylorus screw bacterium fenestra element having adhesion function - Google Patents
Pylorus screw bacterium fenestra element having adhesion function Download PDFInfo
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- CN100378223C CN100378223C CNB03109886XA CN03109886A CN100378223C CN 100378223 C CN100378223 C CN 100378223C CN B03109886X A CNB03109886X A CN B03109886XA CN 03109886 A CN03109886 A CN 03109886A CN 100378223 C CN100378223 C CN 100378223C
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Abstract
The present invention relates to a helicobacter pylori fenestra element, namely a DNA molecule, having an adhesion function, which is characterized in that the DNA molecule comprises (a) a nucleotide sequence shown in the sequence number 1, (b) a nucleotide sequence corresponding to the sequence in (a) at a range of genetic code degeneracy, or (c) a nucleotide sequence hybridized with the sequences in (a) and/or (b) under strict conditions. The present invention has the advantages that gene recombination is used for cloning a helicobacter pylori fenestra element gene with the adhesion function, a vector is constructed to carry out sequence analysis and protein expression on the gene, the adhension function is increased, and helicobacter pylori is prevented and cured through immunoprotection.
Description
Technical field
The present invention relates to a kind of helicobacter pylori fenestra element with adhesion function.
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.
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.But, relevant this adhesin but is controversial to the meaning of helicobacter pylori infection.Other potential adhesin or only identify by its molecular weight or according to its receptors bind specificity.They comprise a kind of 63kDa albumen, it as if with the extracellular enzyme S homology of Pseudomonas aeruginosa (Pseudomonasaeruginosa), be a kind of adhesin of the ADP-of having ribosyl-transferase active.Also suspecting has a kind of adhesin that does not identify as yet, and it can mediate the Lewis with gastric epithelial cell
bThe specificity of blood group antigen is in conjunction with (Falk etc., 1993, Boren etc., 1993).
Be subjected to helicobacter pylori infection can cause the chronic inflammatory reaction (gastritis) of stomach mucous membrane.In addition, bring out specificity systemic immune response to Heliobacter pylori antigen; But, the formation under one's belt of secretion property antibody (slgA) does not obtain incontrovertible elaboration as yet.There is the panimmunity cell in the result of inflammation in stomach mucous membrane and inferior mucous membrane, for example polymorphonuclear leukocyte, monocyte, scavenger cell, lymphocyte and plasmocyte (Blaser, 1992).In addition, helicobacter pylori is in external activation neutrophil, monocyte and scavenger cell (Mai etc., 1991).The experiment of carrying out with specific antibody and complement shows, makes the rapid inactivation of helicobacter pylori at external neutrophil.
Summary of the invention
Purpose of the present invention is exactly at above-mentioned the deficiencies in the prior art part; a kind of helicobacter pylori fenestra plain gene that has adhesion function with the gene recombination clone is provided; and carrier construction carries out sequential analysis and protein expression analysis to it, the helicobacter pylori fenestra element with adhesion function of raising adhesion function and immunoprotection.
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, and (c) the cell suppressed by vector transforms; And (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 fenestra plain 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.
The protein electrophoresis analytical results is found, efficiently express relative molecular weight and be 56500 albumen after inducing, the gel autoscan is analyzed, and its reorganization adhesin conserved regions albumen accounts for 31.9% of bacterial protein, wherein solubility expression accounts for 23.7% of supernatant, and inclusion body accounts for sedimentary 64.8%.
Externally stick antiserum(antisera) that experiment shows that this fenestra element with adhesion function is positioned at bacterium surface and its fusion rotein and can stop helicobacter pylori fully, and the sero-fast result of its control experiment urease B fusion rotein in contrast the sticking of stomach-tissue.In addition, animal shows that this fenestra element with adhesion function of helicobacter pylori can be used for prevention and treatment helicobacter pylori infection.
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-22b of the present invention (+)/fenestra element are identified collection of illustrative plates,
Fig. 3 is the SDS-PAGE analysis chart of the plain recombinant protein of fenestra of the present invention,
Fig. 4 is a sequence number 1 of the present invention.
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, adhesive agent 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 gene clone technology that the gene of the plain sample adhesin of fenestra has been carried out the clone and expressed and studied its immune protection effect.
1 materials and methods
1.1 plasmid and bacterial strain
Bacterial strain BL21 (DE3), plasmid pET-22b (+) and helicobacter pylori SS1 preserve for digestion institute of Nanfang Hospital of No.1 Military Medical Univ..
1.2 toolenzyme and reagent
Restriction enzyme Not I, Noc I and T4DNA polysaccharase, Vent archaeal dna polymerase are available from New EnglandBiolabs company, Taq archaeal dna polymerase, 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 U.S. Qiagen company, and other reagent is homemade analytical pure.
1.3Hp the extraction of chromosomal DNA
Scrape from solid medium and to 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 of plasmid and purifying
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
The design primer adds suitable restriction enzyme site at its 5 ' end, and is synthetic by Bo Ya company.Sequence is as follows: the plain sample adhesin of fenestra 1:5 '-TG G
CC ATG GAT TGC GCT AGC ATA AGT TA-3 ' Nco I
The plain sample adhesin of fenestra 2:5 '-AG T
GC GGC CGCGAA 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 50s, 72 ℃ are extended 90s, extend 10min again after 35 circulations.0.8% agarose gel electrophoresis is observed amplification.
1.6DNA segmental enzyme is cut, is connected, the evaluation of conversion and positive colony
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 sequential analysis with automatic sequencer.
1.8 abduction delivering and SDS-polyacrylamide gel electrophoresis
The plain sample adhesin of fenestra positive colony carries out the SDS-polyacrylamide gel electrophoresis behind the IPTG abduction delivering.
1.9 experimentation on animals
Give fenestra plain sample adhesin to the mouse that infects helicobacter pylori by the mucous membrane approach, observe therapeutic action.Give fenestra plain sample adhesin by the mucous membrane approach earlier to the mouse that does not infect helicobacter pylori, and then attack, observe provide protection with helicobacter pylori.
1.10 light microscopic quantitative counting method is measured adhesive activity
The MGC-803 cell inoculation is cultured in 6 orifice plates of slide with cover and forms monolayer, takes out cover glass, after the PBS rinsing 1 time with positive clone strain or empty carrier clone strain suspension (10
9CFU/ml) drip to cover glass, hatch 40min in 37 ℃ of wet boxes, do not adhere to bacterium to remove with PBS rinsing 6 times, seasoning, fixing, Gram dyeing.The oil mirror is observed the adhesive attraction of MGC-803 and bacterium down, calculates the adherent bacterial count of each cell peripheral, repeats 3 times, averages, and every part of sample is counted 30 cells altogether at random, and the result represents with mean ± standard deviation.
1.11 statistical procedures
Adopt the independent sample t check in the SPSS7.0 software.There is the significance meaning P＜0.01 for difference.
2 results
2.1 the amplification of the plain sample adhesin of fenestra
PCR electrophoretic analysis as a result finds that a band is arranged about 1500bp, size fulfills the expectation, and the results are shown in Figure 1.
2.2 construction of recombinant plasmid and enzyme are cut evaluation
Behind Not I and Noc I double digestion, the directed insertion in pET-22b (+) carrier of same double digestion obtains the plain sample adhesin of recombinant plasmid called after pET-22b (+)/fenestra with the PCR product.Recombinant plasmid electrophoresis behind Not I and Noc I double digestion the results are shown in Figure 2.
2.3 the plain sample adhesin gene of fenestra fragments sequence is analyzed
Be that template checks order with the plain sample adhesin of recombinant plasmid pET-22b (+)/fenestra directly, obtained the dna sequence dna of cloned sequence, the automatic sequencer The sequencing results is seen below (Fig. 4).
2.4 the expression of the plain sample adhesin gene of fenestra in intestinal bacteria
With the plain sample adhesin positive clone strain of fenestra 37 ℃ of incubated overnight in LB nutrient solution (containing the 100ug/ml penbritin), be forwarded in the LB nutrient solution that contains penbritin by 1% then, continuing to be cultured to the D value is 0.6～0.8, add IPTG to final concentration be 0.1mmol/L, abduction delivering 3h, centrifugal collection thalline is got osmotic shock liquid, the thalline of its pericentral siphon supernatant and the precipitation after ultrasonic and is carried out 10%SDS-PAGE electrophoretic analysis (Fig. 3).Found that expressing relative molecular weight after inducing is 56.5 * 10
3
The plain sample adhesin of fenestra recombinant protein accounts for 31.9% of bacterial protein, and wherein solubility expression accounts for 23.7% of supernatant, and inclusion body partly accounts for sedimentary 64.8%.
2.5 experimentation on animals
The treatment of the plain sample adhesin of fenestra and protect and efficiently be 100%.
2.6 light microscopic sticks experimental result
Compare with empty carrier clone strain treatment group, positive clone strain treatment group cell peripheral bacterial count showed increased (P＜0.01), its difference has significance.Light microscopic sticks experimental result and shows that the plain sample adhesin of fenestra has the effect of sticking.
Reference
[1]Sambrook?J，Fritsch?EF，Maniatis?T.Molecular?cloning：a?laboratory?manua?1.2
nd?ed.New?York：Cold?Spring?Harbor?Laboratory?Press，1989.35.
[2] Ausubel FM, Brent R, Kingston RE, et al. face grain husk, Wang Hailin is translated.Fine works molecular biology guide.Beijing: Science Press, 1998.39.
Claims (4)
1.DNA molecule is characterized in that, it is selected from:
(a) nucleotide sequence as shown in Figure 4,
(b) with Fig. 4 in the nucleotide sequence of nucleotide sequence with genetic code degeneracy.
2. comprise the cell of the dna molecular of claim 1, it is characterized in that:
(a) carrier that wherein includes described dna molecular contains this dna molecular of at least one copy,
(b) with (a) described carrier transformant.
3. polypeptide is characterized in that, it is coded by the dna molecular of claim 1.
4. produce the method for claim 3 polypeptide, under the condition that polypeptide is expressed, the cell of the described polypeptide of culture expression claim 3, and from cell or/and isolate polypeptide the culture supernatants.
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| CNB03109886XA CN100378223C (en) | 2003-04-17 | 2003-04-17 | Pylorus screw bacterium fenestra element having adhesion function |
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| CNB03109886XA CN100378223C (en) | 2003-04-17 | 2003-04-17 | Pylorus screw bacterium fenestra element having adhesion function |
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| CN100378223C true CN100378223C (en) | 2008-04-02 |
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Citations (5)
| 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 |
| WO1999049890A1 (en) * | 1998-03-31 | 1999-10-07 | Daewoong Pharmaceutical Co., Ltd. | A preventive and therapeutic vaccine for helicobacter pylori-associated diseases |
| WO2001010386A2 (en) * | 1999-08-11 | 2001-02-15 | Washington University | Anti-bacterial compounds directed against pilus biogenesis, adhesion and activity; co-crystals of pilus subunits and methods of use thereof |
| CN1301571A (en) * | 1999-12-24 | 2001-07-04 | 南方医院 | Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine |
| WO2002064622A1 (en) * | 2001-02-05 | 2002-08-22 | Merieux Oravax | Method for purifying the helicobacter adhesin-like protein a (alpa) |
-
2003
- 2003-04-17 CN CNB03109886XA patent/CN100378223C/en not_active Expired - Fee Related
Patent Citations (5)
| 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 |
| WO1999049890A1 (en) * | 1998-03-31 | 1999-10-07 | Daewoong Pharmaceutical Co., Ltd. | A preventive and therapeutic vaccine for helicobacter pylori-associated diseases |
| WO2001010386A2 (en) * | 1999-08-11 | 2001-02-15 | Washington University | Anti-bacterial compounds directed against pilus biogenesis, adhesion and activity; co-crystals of pilus subunits and methods of use thereof |
| CN1301571A (en) * | 1999-12-24 | 2001-07-04 | 南方医院 | Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine |
| WO2002064622A1 (en) * | 2001-02-05 | 2002-08-22 | Merieux Oravax | Method for purifying the helicobacter adhesin-like protein a (alpa) |
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