CN1331876C - Recombinant helicobacter pylori blood group antigen adhesin - Google Patents
Recombinant helicobacter pylori blood group antigen adhesin Download PDFInfo
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- CN1331876C CN1331876C CNB031098886A CN03109888A CN1331876C CN 1331876 C CN1331876 C CN 1331876C CN B031098886 A CNB031098886 A CN B031098886A CN 03109888 A CN03109888 A CN 03109888A CN 1331876 C CN1331876 C CN 1331876C
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Abstract
The present invention relates to recombinant helicobacter pylori blood group antigen adhesin. A DNA molecule is characterized in that the DNA molecule comprises (a), a nucleotide sequence shown in a serial number 1, (b), a nucleotide sequence corresponding to the sequence in (a) within range of the degeneracy of a genetic code, or (c), a nucleotide sequence crossed with the sequence/sequences in (a) or/and (b) under the stringent condition. The present invention has the advantages that a PCR technology is utilized to clone a gene of a blood group antigen of helicobacter pylori and builds a carrier and analyzes the sequence and the protein expression of the carrier; increasing antiserum can prevent the helicobacter pylori from being adhered to the epithelium of the gastric mucosa.
Description
Technical field
The present invention relates to the helicobacter pylori adhesin, particularly a kind of recombinant helicobacterpylori blood group antigen adhesin.
Background technology
For a long time, there is spirillar bacterium (Bizzozero, 1893) in people's known person stomach mucous membrane.But, when Marshall and Warren successfully isolated from stomach ulcer patient's stomach mucous membrane and cultivate this bacterium, people recognized that just they are this fact of pathogenic bacteria (Warren and Marshall, 1983; Marshall etc., 1984).Initial analysis revealed, isolated microorganism are the negative spirillar bacteriums of the blue people of leather, and it has high motility and can (reach pH1.5 approximately) under strong acidic condition the outstanding ability of survival.These bacteriums that are called as campylobacter pylori (Campylobacterpylori) at first finally are included into " Helicobacter pylori (the Helicobacter) " genus (Goodwin etc., 1989) of new establishment according to biochemical and morphological specificity.
In the past few years, the epidemiological study of being done from Taylor and Blaser (1991) shows that helicobacter pylori infection takes place in the world, has 50% population to be subjected to this infectation of bacteria approximately, and developing country is higher than the infection rate of industrialized country.And with the raising at age, the probability of chronic helicobacter pylori infection sharply increases.Therefore, chronic helicobacter pylori infection is one of human modal chronic bacterial infection.
Know that at present this infection causes bringing out of human bacterial gastritis (Type B gastritis) inevitably.In addition, suppose that also helicobacter pylori plays inducement effect (Lee etc., 1993 in the development of the cancer of the stomach (gland cancer) of stomach ulcer and duodenal ulcer and some form; Solnick and Tompkins, 1993).More rare stomach MALT (mucosa associated lymphoid tissue) lymphoma that is regarded as the tendency of immunity system B glucagonoma probably also is the result of helicobacter pylori infection.The antibacterial therapy of the successful eradicate helicobacter pylori of energy that this class patient is carried out causes stomach ulcer and the lymphadenomatous healing of rudimentary MALT (Spponen and Hyvarinen, 1993; Isaacson and Spencer, 1993; Stolte and Eidt, 1993).
Being subjected to a consequence of helicobacter pylori infection for a long time is atrophic gastritis, viscosity, produces acid or produces pepsic gastric epithelial cell sex change, is regarded as lesion precancerous.According to the statistics to the common cancer type in the whole world in 1980, cancer of the stomach occupied the second, but downtrending (Parkin etc., 1988) is arranged.Studies show that the statistics significant correlation between (visible peristalsis visible intestinal peristalsis) takes place for helicobacter pylori infection and cancer of the stomach; The conclusion that both draw all is, has 60% in whole cancer of the stomach of generation approximately the chances are result (Parsonnet etc., 1991 of helicobacter pylori infection; Nomura etc., 1991).What Sipponen (1992) was done further studies show that, the people who is infected in many industrialized countries has more than 20% and suffers from stomach ulcer or duodenal ulcer in life at it; And for the normal people of stomach mucous membrane, this danger is little as can to ignore.Common stomach-the dudenal disease of this class must be regarded as transmissible disease and need suitably treatment (Alper, 1993).Eliminate a therapy of the chronic helicobacter pylori door helicobacter infection that has taken place and can cure gastritis, stomach ulcer or duodenal ulcer or MALT lymphoma.Therefore, can adopt preventative therapy (for example, immunization) that prevents helicobacter pylori infection and the therapy of eliminating the helicobacter pylori infection that has taken place to treat the common stomach-dudenal disease of this class.
Bacterium at first arrives emitting in the chamber (pH1-2) of pinch acid after taking in from mouth.At this, by producing the urase this kind of enzyme bacterium may be survived, described urase causes the decomposition of existing urea, thereby causes the part neutralization of acid ph value in the stomach.By chemotactic orientation and the motility that depends on flagellum, microorganism moves to the mucous layer of the buffered with bicarbonate of stomach Dou Qu again, and they are herein in the ecological niche of uniqueness.Because the acid barrier effect has only several competitive bacterias can enter this ecotope.For arriving epithelium, microorganism probably carries out self orientation by means of the pH gradient between chamber (pH1-2) and the surface epithelial cell (Ph6-7).Because the generation and the microaerophilic mode of life thereof of its spiral shell shape, its motility at mucous membrane, mucous membrane modifying enzyme, these bacteriums have adapted to the living condition in this site best.
They are lived in the dark gland (deep crypts) of Dou Qu usually, avoid as acid, ectocine the stomach en-at this, also avoid medicine as microbiotic to its removing of carrying out.Part flora (about 20%) and epithelial cell, particularly to produce myxocyte closely related.Under the epitheliogenic condition of stomach of promptly being brought out by acid in duodenum under the condition that stomachization is given birth to, the metaplastic region in the duodenum is also moved life; This has just produced the prerequisite of duodenal ulcer development.Discharge the obstruction that probably is subjected to its adhesive capacity fully with effusive mucus, bacterium just can survive several years, decades or even all one's life (chronic infection) like this.
Before the existence of knowing helicobacter pylori and its meaning, treat with so-called antacid or H2-receptor antagonist to Peptic Ulcers.Though can suppress parietal cell secretion acid, can cure ulcer usually, can not eliminate Peptic Ulcers thus.
Ulcer therapy more commonly used is that bismuth is handled.Various bismuth salt (CBS, BSS) all have sterilization effect to helicobacter pylori.But only in the case of 8-32%, reach the effect of removing bacterium fully.Cause the temporary transient inhibition of bacterium on this treat surface, the infection meeting shows effect once more in the most cases in back but stop to handle.Cause the accumulation of these materials in liver, kidney and neural system with high dosage long-term treatment meeting, have sizable neuroscience side effect (Malfertheiner, 1994).
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, 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 in external activation neutrophilia from cell, 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, provide a kind of utilization pcr clone helicobacter pylori blood group antigen gene and carrier construction that it is carried out sequential analysis and protein expression analysis, improve antiserum(antisera) and can stop helicobacter pylori to adhere to the recombinant helicobacterpylori blood group antigen adhesin 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 with (a) or/and the nucleotide sequence of sequence hybridization (b): and (a) it the coding a kind of polypeptide that can adhere to people's cell, (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, at least 98% homology most preferably.
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 protein electrophoresis analytical results is found, after inducing, efficiently express relative molecular weight and be 78000 albumen, the gel autoscan is analyzed, its reorganization adhesin blood group antigen albumen accounts for 34.8% of bacterial protein, wherein secreting, expressing accounts for 22.7% of pericentral siphon total protein, solubility expression accounts for 15.0% of supernatant, and inclusion body accounts for sedimentary 86.7%.
Experimentation on animals shows that reorganization adhesin blood group antigen albumen is a kind of neoantigen of preparation Hp vaccine.Simultaneously, the external antiserum(antisera) that sticks experiment confirm reorganization adhesin blood group antigen can stop helicobacter pylori to attach to gastric epithelial cells.
The present invention has utilization pcr clone helicobacter pylori blood group antigen 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 advantage such as immunogenicity.
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 (+)/blood group antigen adhesin are identified collection of illustrative plates,
Fig. 3 is the SDS-PAGE analysis chart of blood group antigen adhesin recombinant protein of the present invention,
Fig. 4 is sequence table.
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 gene clone technology that the gene of blood group antigen adhesin 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 film solid media 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.Synthetic by Bo Ya company.
Sequence is as follows:
Blood group antigen adhesin 1:5 '-TG G
CC ATG GAT AAA AAA CAC ATC CTT TCA-3 '
Nco?I
Blood group antigen adhesin 2:5 '-AG T
GC GGC CGC ATA AGC GAA CAC ATA G-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 Not 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
Blood group antigen adhesin positive colony carries out the SDS-polyacrylamide gel electrophoresis behind the IPTG abduction delivering.
1.9 experimentation on animals
Give the blood group antigen adhesin to the mouse that infects helicobacter pylori by the mucous membrane approach, observe therapeutic action.Earlier give the blood group antigen adhesin to the mouse that does not infect helicobacter pylori, and then attack, observe provide protection with helicobacter pylori by the mucous membrane approach.
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, seasoning to remove with PBS rinsing 6 times, fixing, the adhesive attraction that MGC-803 and bacterium are observed in Gram dyeing, oily mirror down calculates the adherent bacterial count of each cell peripheral, repeat 3 times, average, 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 blood group antigen adhesin gene
PCR electrophoretic analysis as a result finds that a band is arranged about 2200bp, 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 recombinant plasmid called after pET-22b (+)/blood group antigen adhesin with the PCR product.Recombinant plasmid electrophoresis behind Not I and Noc I double digestion the results are shown in Figure 2.
2.3 blood group antigen adhesin gene fragments sequence is analyzed
Be that template checks order with recombinant plasmid pET-22b (+)/blood group antigen adhesin 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 blood group antigen adhesin gene in intestinal bacteria
With blood group antigen adhesin positive clone strain 37 ℃ of incubated overnight in LB nutrient solution (containing 100ug/ml ammonia benzyl perimycin), press 1%mmol/L then, 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, after inducing, efficiently express the albumen that relative molecular weight is 78kD, consistent with expection molecular weight size, the gel autoscan is analyzed, its reorganization blood group antigen adhesin albumen accounts for 34.8% of bacterial protein, wherein secreting, expressing accounts for 22.7% of pericentral siphon total protein, and solubility expression accounts for 15.0% of supernatant, and inclusion body accounts for sedimentary 86.7%.
2.5 experimentation on animals
The treatment of blood group antigen adhesin and protect and efficiently be 100%.
2.6 light microscopic sticks the experiment structure
Compare with empty carrier clone strain treatment group, and 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 blood group antigen adhesin has the effect of sticking.
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 (9)
1, dna molecular 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).
2, dna molecular as claimed in claim 1 is characterized in that on nucleic acid level nucleotides sequence is shown at least 90% homology shown in it and the sequence number 1.
3, claim 1 or 2 dna molecular is characterized in that its length is at least 45 Nucleotide.
4, the dna molecular of claim 3 is characterized in that
(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.
5, polypeptide is characterized in that it is coded by the dna molecular of one of claim 1～4.
6, the polypeptide of claim 5 is characterized in that 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.
7, produce the method for the polypeptide of one of claim 5～6, it is characterized in that transforming a kind of cell with the dna molecular of one of claim 1～3 or the carrier of claim 4, under the condition that polypeptide is expressed, cultivate cell transformed, and from cell or/and isolate polypeptide the culture supernatants.
8, the polypeptide of one of claim 5～6 produces the application of antibody and the antibody of polypeptide as immunogen.
9, medicinal compositions is characterized in that:
(a) it comprises the polypeptide of one of dna molecular, claim 5～6 of one of claim 1～3 as active substance or the antibody of claim 8, optionally comprises common medical aid matter, thinner, additive and carrier.
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| CN1301571A (en) * | 1999-12-24 | 2001-07-04 | 南方医院 | Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1301571A (en) * | 1999-12-24 | 2001-07-04 | 南方医院 | Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine |
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