CN1301571A - Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine - Google Patents
Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine Download PDFInfo
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- CN1301571A CN1301571A CN 99124665 CN99124665A CN1301571A CN 1301571 A CN1301571 A CN 1301571A CN 99124665 CN99124665 CN 99124665 CN 99124665 A CN99124665 A CN 99124665A CN 1301571 A CN1301571 A CN 1301571A
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- hpaa
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- helicobacter pylori
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- adhesin hpaa
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Abstract
The present invention features that the microcapsulated helicobacillus pyloricus adherent agent (HpaA) with nucosa is fed to an animal, and has obvious immunogenicity. The present invention has simple technolgicy process and high immunological, effect, and it may be used as an important and effective remedy for treating chronic aastritis, peptic ulcer and even gastric cancer
Description
The present invention relates to a kind of method of production of vaccine, particularly the method for production of helicobacter pylori adhesin HpaA recombinant vaccine.
Helicobacter pylori (Helicobacter pylori, Hp) be one of human modal chronic infection bacterium, it is the main pathogen factor of chronic active gastritis and peptic ulcer, also closely related with adenocarcinoma of stomach and the lymphadenomatous generation of gastric mucosa dependency lymphoid tissue (MALT), cancer research mechanism of World Health Organization (WHO) (IARC) has classified it as the 1st class cancerigenic factor.To effective prevention and the treatment that Hp infects, may make relevant disease such as peptic ulcer become historic disease.The means of existing elimination Hp mainly are use in conjunction acid inhibitor and two or three kind of antibiotic, but because the universality (especially developing country) that Hp infects, the continuous increase of Resistant strain, patient's the low compliance and the factors such as costliness of combined treatment, make antimicrobial therapy be difficult to become the best approach of eradicating this bacterium fully, if can successfully develop the medicative again vaccine of a kind of existing prophylactic function can make numerous Hp the infecteds be benefited undoubtedly, and may prevent millions of routine ulcer, the generation of lymphoma and adenocarcinoma of stomach brings huge social and economic benefit.
Abroad from pioneer's research of beginning Hp vaccine in 1991, the immunogen of Cai Yonging is full bacterium lysate the earliest, afterwards because of complicated component, have potential toxic and side effects and gradually eliminated; Ideal protective antigen should high-purity, avirulence, easily preparation, and can disturb the field planting of antibacterial and the generation of virulence, and gene recombinant protein becomes inevitable choice because of possessing above-mentioned advantage.The immunogen that has adopted at present mainly contains urease (Urease), cell ghost poison pure (VacA) and toxin associated protein (CagA), heat shock protein (HspA/HspB), catalase etc., and they all can protect mice more than 50% to avoid the infection of Helicobacter pylori under the combined effect of mucosal adjuvants.But a kind of antigen obviously unlikely induces enough protective immunities at human body, allow all experimenters all produce effective protective effect, and enough target antigens must be arranged.Existing scholar confirms that the vaccine that is combined by two kinds of antigens has than the stronger protective effect of both independent uses.Almost the vaccine that can be combined by two or more antigen certainly is essential.
U.S. Oravax company and Switzerland Michetti group have cooperated to finish the I clinical trial phase of recombinant urease immunization therapy effect.Discovery is under adjuvant participates in, oral recombinant urease can make bacteria planting density descend significantly, but inflammation or mucosa injury degree almost do not change, also non-evident effect produces, further confirm the feasibility that immunity inoculation control Hp infects, but be also shown in need in the use of immunogenic selection, immunization route and vaccine delivery mode further perfect.
As described above, although existing at present multiple antigen component is estimated in animal model, they are conceived to block the virulence factor of Hp basically, and do not estimate as yet with the closely-related adhesin of Hp field planting.According to the research experience of the past enteric infection disease vaccine, blocking-up colonizing factor and virulence factor are the two big links that vaccine is had an effect, and both are indispensable.
Purpose of the present invention be intended to overcome the deficiencies in the prior art and provide that a kind of technology is simple and direct, good immune effect to be to obtain the method for production of helicobacter pylori adhesin HpaA recombinant vaccine.
The object of the present invention is achieved like this:
A kind of method of production of helicobacter pylori adhesin HpaA recombinant vaccine is characterized in that being aided with mucosal adjuvants with adhesin HpaA, gavages animal again behind the microcapsule parcel, has tangible immunogenicity.
---described adhesin HpaA is through clone and expression, with concentration is that the ammonium sulfate precipitation of 30-40% concentrates and slightly to carry, reuse solution A (the 25m μ TvisCl of PH8.0～8.2) and solution B (A+1MNaCl) are carried out anion-exchange chromatography, carry out gel permeation chromatography through SephodexG100FF again, reached HpaA recombiant protein more than 98% after ultrafiltration and concentration has both made purity.
---described mucosal adjuvants is good with LTK63.
---described microcapsule parcel is good with diameter 6～8 μ m.
---described animal is good with SPF level BalB/C mice.
The invention will be further described below by embodiment:
After the HpaA pure protein that the technology of by specification makes is aided with mucosal adjuvants LTK63; make the microcapsule of diameter 6～8 μ m; to SPF level BalB/C mice gavage the 1st day, the 3rd day and the 5th day; by the time after using the HP viable bacteria to attack in the time of the 20th day, the 23rd day, the 25th day; put to death animal during to 2 months, 5 months in batches; detect the field planting situation of mice gastric mucosa HP, to determine protective rate.
The result is: fully protective rate is 40% (40% mice does not infect HP), and all the other mice antibacterial bacterium of 60% amounts obviously reduce than matched group, and single mice with PBS or single matched group 100% with the LTK63 adjuvant all infects HP.
Method of production of the present invention adopt with the closely-related adhesin HpaA of HP field planting be immunogen, the acquisition of this recombinant antigen is for preparing multivalence in the future or multicomponent HP vaccine provides an important alternative antigen.Simple and direct, the good immune effect of technology of the present invention, its pharmaceutical preparation will provide the important and effective ways of treatment chronic gastritis, peptic ulcer and even gastric cancer etc.
Claims (5)
1, a kind of method of production of helicobacter pylori adhesin HpaA recombinant vaccine is characterized in that being aided with mucosal adjuvants with adhesin HpaA, gavages animal again behind the microcapsule parcel, has tangible immunogenicity.
2, the method for production of helicobacter pylori adhesin HpaA recombinant vaccine according to claim 1, it is characterized in that adhesin HpaA is through clone and expression, with concentration is that the ammonium sulfate precipitation of 30-40% concentrates and slightly to carry, reuse solution A (the 25m μ TvisCl of PH8.0～8.2) and solution B (A+1MNaCl) are carried out anion-exchange chromatography, carry out gel permeation chromatography through SephodexG100FF again, reached HpaA recombiant protein more than 98% after ultrafiltration and concentration has both made purity.
3, the method for production of helicobacter pylori adhesin HpaA recombinant vaccine according to claim 1 is characterized in that mucosal adjuvants is good with LTK63.
4, the method for production of helicobacter pylori adhesin HpaA recombinant vaccine according to claim 1 is characterized in that the microcapsule parcel is good with diameter 6～8 μ m.
5, the method for production of helicobacter pylori adhesin HpaA recombinant vaccine according to claim 1 is characterized in that animal is good with SPF level BalB/C mice.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99124665 CN1301571A (en) | 1999-12-24 | 1999-12-24 | Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99124665 CN1301571A (en) | 1999-12-24 | 1999-12-24 | Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1301571A true CN1301571A (en) | 2001-07-04 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99124665 Pending CN1301571A (en) | 1999-12-24 | 1999-12-24 | Development method for pylorus helicobacterium adhesion agent HpaA gene engineering vaccine |
Country Status (1)
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| CN (1) | CN1301571A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331876C (en) * | 2002-04-11 | 2007-08-15 | 南方医院 | Recombinant helicobacter pylori blood group antigen adhesin |
| CN100378223C (en) * | 2003-04-17 | 2008-04-02 | 南方医院 | Helicobacter pylori membrane porin with adhesion function |
| CN102796757A (en) * | 2012-06-28 | 2012-11-28 | 郑州大学 | Carrier capable of showing and expressing helicobacter pylori protein on surface of lactococcus lactis, and preparation method and application of carrier |
-
1999
- 1999-12-24 CN CN 99124665 patent/CN1301571A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331876C (en) * | 2002-04-11 | 2007-08-15 | 南方医院 | Recombinant helicobacter pylori blood group antigen adhesin |
| CN100378223C (en) * | 2003-04-17 | 2008-04-02 | 南方医院 | Helicobacter pylori membrane porin with adhesion function |
| CN102796757A (en) * | 2012-06-28 | 2012-11-28 | 郑州大学 | Carrier capable of showing and expressing helicobacter pylori protein on surface of lactococcus lactis, and preparation method and application of carrier |
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