Constructing genetic engineering Vaccine of adhesin of confluent Helicobacter pylor and preparation method

A genetically engineered vaccine, Helicobacter pylori technology, applied in the direction of peptide preparation methods, genetic engineering, botany equipment and methods, etc., can solve the complex anti-Hp treatment, can not effectively prevent Hp reinfection and recurrence, single-dose drug treatment low eradication rate

Inactive Publication Date: 2006-05-10
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
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  • Application Information

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Problems solved by technology

There are still big problems in the anti-infection treatment of Hp. The eradication rate of single drug therapy is low, while the use of multiple drugs is expensive, the cure rate is not high, and it cannot effectively prevent Hp reinfection and recurrence. In addition, the increase of drug-resistant strains is also increasing. Make anti-Hp therapy face more and more complicated problems

Method used

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  • Constructing genetic engineering Vaccine of adhesin of confluent Helicobacter pylor and preparation method
  • Constructing genetic engineering Vaccine of adhesin of confluent Helicobacter pylor and preparation method
  • Constructing genetic engineering Vaccine of adhesin of confluent Helicobacter pylor and preparation method

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1, construction and preparation method of LTB / HpaA genetic engineering vaccine

[0046] 1. Construction of genetically engineered bacteria

[0047] 1. Primer design and synthesis

[0048] Using wild-type toxigenic Escherichia coli H44815 (purchased from China National Institute for the Control of Pharmaceutical and Biological Products, stored in our laboratory), Helicobacter pylori SS1 (Hp Sydney strain, named by Lee A in Australia, etc., stored in our laboratory) as templates, use P1 and P2, P5 and P6 to amplify the LTB and HpaA genes, and the bacterial genome was extracted according to conventional methods (Yan Ziying, translated by Wang Hailin. Refined Guide to Molecular Biology Experiments. Science Press, 1998, P39). The PCR amplification system is: 10 μL of amplification buffer without magnesium ions, 10 μL of MgCl 2 (25mmol / L) 10μL, dNTPs (2.5mmol / Leach) 8μL, upstream and downstream primers (P1 and P2 or P5 and P6) 2μL each, the above LTB genome 2μL, ...

Embodiment 2

[0062] Embodiment two, the construction and preparation method of CTB / HpaA genetic engineering vaccine

[0063] 1. Construction of genetically engineered bacteria

[0064] 1. Primer design and synthesis

[0065] Using Escherichia coli 0139 (from the American Type Culture Collection, ATCC31165) and Helicobacter pylori SS1 genomic DNA as templates respectively, CTB and HpaA genes were amplified with P3 and P4, P5 and P6, and the genome extraction method was the same as in Example 1 . The PCR amplification system was: 10 μL of 10× Mg+ ion-free amplification buffer, 10 μL of MgCl2 (25 mmol / L), 8 μL of dNTPs (2.5 mmol / L each), 2 μL of upstream and downstream primers (P1 and P2), and the above CTB genome 2 μL, Ex-Taq DNA polymerase (3 units / μL) 1 μL, add sterilized water to 100 μL.

[0066] PCR amplification reaction: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 50 s, annealing at 60°C for 50 s, extension at 72°C for 50 s, 35 cycles, and complete extension at 72°C...

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Abstract

The invention discloses the construction and preparation method of fusion type Helicobacter pylori adhesin genetic engineering bacteria vaccine, which is characterized in that the adhesin HpaA is used to construct the Helicobacter pylori adhesin and Escherichia coli heat-labile enterotoxin B subunit or cholera toxin B subunit intramolecular adjuvant fusion engineered bacteria, through high-density fermentation, inclusion body extraction, renaturation and purification to obtain a large amount of required vaccine protein, the vaccine can be prepared into oral preparations, injections and drops / nasal spray. The construction method of the genetic engineering vaccine is unique, the preparation process is simple, easy to scale up, and has good repeatability. The obtained vaccine protein has high purity. Animal experiments have proved that it can effectively stimulate the body to produce a higher immune response and immune protection.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to the construction and preparation method of fusion type Helicobacter pylori adhesin genetic engineering vaccine Background technique [0002] Helicobacter pylori (Hp) is a Gram-negative Helicobacter that colonizes the human gastric mucosa, and is one of the most common pathogenic microorganisms that cause chronic infection. More than 50% of people in the world have been infected with Hp. Most infected people usually have no symptoms, but about 30% of infected people develop chronic gastritis, 10-20% develop peptic ulcer (gastric ulcer and duodenal ulcer), and some infected people Gastric mucosal atrophy and intestinal metaplasia can occur on the basis of chronic active gastritis, and a few of them can develop gastric cancer. There are still big problems in the anti-infection treatment of Hp. The eradication rate of single drug therapy is low, while the use of multipl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/31C12N15/63C12N15/11C12N15/09C12N15/70C07K1/14C12P21/02A61K38/16A61P1/04A61P31/04
Inventor 邹全明刘正祥洪愉朱永红童文德解庆华
Owner ARMY MEDICAL UNIV
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