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Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process

A technology of Helicobacter pylori and active fragments, applied in the field of genetic engineering multivalent subunit vaccines and its preparation, can solve the problems of fusion protein refolding difficulty, fusion protein expression rate low, fusion protein purification difficulty, etc., and achieve good immune protection The effect of action

Inactive Publication Date: 2007-01-03
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present inventors found in previous experimental studies that the use of a fusion protein (more than 130KD in molecular weight) comprising a full-length urease B subunit as an immunogen to prepare a vaccine will mainly have the following problems: (1) the expression rate of the fusion protein is low; ( 2) Difficulty in purification of fusion protein; (3) Difficulty in renaturation and low activity of fusion protein

Method used

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  • Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process
  • Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process
  • Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Screening of Helicobacter pylori urease B active fragments

[0052] Using the http: / / cubic.bioc.columbia.edu / predictprotein protein online tool, the functional region of UreB protein and the secondary structure such as hydrophilicity, surface probability, and antigenicity of the full sequence protein were analyzed. Combined with the tertiary conformation of UreB protein X-ray crystallography, select an active fragment with a relatively stable structure and a functionally active domain, that is, the 138 amino acid sequence from the 251st amino acid to the 389th amino acid at the 5' end of the UreB protein (named UreB414) , to replace the full-length UreB protein. The nucleotide sequence of UreB414 shown in SEQ ID NO:1, and the protein structure is the amino acid sequence of UreB414 shown in SEQ ID NO:2.

Embodiment 2

[0053] Example 2 Preparation of recombinant antigenic protein, recombinant adjuvant protein and recombinant fusion protein

[0054] 1 Cloning the coding genes of HspA, HpaA, UreB414 and LTB of Helicobacter pylori according to conventional methods.

[0055] 2 Construct UreB414-LTB, HspA-UreB414, HpaA-UreB414, HspA-UreB414-LTB, HpaA-UreB414-LTB, and HspA-HpaA-UreB414 fusion genes respectively according to the following steps.

[0056] a) The fusion genes of UreB414-LTB, HspA-UreB414, HpaA-UreB414, HspA-UreB414-LTB, HpaA-UreB414-LTB, and HspA-HpaA-UreB414 were obtained by the overlap extension method, and a linker sequence was introduced between each gene.

[0057] b) PCR amplification products were separated by agarose gel electrophoresis.

[0058] c) Cloning the recovery products of the fusion genes of UreB414-LTB, HspA-UreB414, HpaA-UreB414, HspA-UreB414-LTB, HpaA-UreB414-LTB, and HspA-HpaA-UreB414 into prokaryotic cell expression vectors to obtain the above-mentioned Recomb...

Embodiment 3

[0065] Example 3 The preparation method of multivalent combination vaccine

[0066] Method 1: Physically mix recombinant UreB414 protein, at least one recombinant antigen protein (recombinant HspA, recombinant HpaA, recombinant Vac, recombinant CagA, recombinant NAP and recombinant Catalase), and recombinant adjuvant protein LTB or CTB in an appropriate proportion , to prepare the Hp multivalent combination vaccine.

[0067] Method 2: Link the nucleic acid sequence of UreB414 with the nucleic acid sequence of at least one recombinant antigen (HspA, HpaA, Vac, CagA, NAP, and Catalase) at the gene level in different combinations to construct a polynucleotide that expresses a combination of different antigens. A recombinant fusion protein, and on this basis, it is physically mixed with LTB or CTB and other adjuvant components according to an appropriate ratio to prepare genetically engineered recombinant multivalent fusion protein vaccines with different extramolecular adjuvants....

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Abstract

The present invention provides one kind of genetic engineering multivalent subunit vaccine for preventing and treating human helicobacter pylori infection and its preparation process. The vaccine consists of active helicobacter pylori UreB fragment UreB414 as the central antigen component, other combined protective antigens, and intramolecular or extramolecular adjuvant. Compared with univalent vaccine, the multivalent combined vaccine can stimulate the body to generate more powerful and more comprehensive helicobacter pylori resisting specific immune reaction.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a genetic engineering multivalent subunit vaccine used for the immune prevention and treatment of human Helicobacter pylori infection and a preparation method thereof. Background technique [0002] Helicobacter pylori (Helicobacter pylori, Hp) is a worldwide human infection pathogenic bacteria, infecting more than half of the world's population. Hp is associated with many diseases such as chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma (MALT) and gastric cancer, and poses a serious hazard to human health. The World Health Organization has listed it as a class of carcinogens. In 1998, Japanese scholar Watanadbe et al. first reported that Hp-infected Mongolian gerbils could induce gastric cancer, providing direct evidence that Hp infection can induce gastric cancer. Drug treatment of Hp has a low eradication rate, is expensive, and cannot effect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/116A61K38/43A61P31/04C12N15/09C12N15/62C12N1/21C12N9/14C07K14/195
Inventor 邹全明杨珺袁小澎柏杨张卫军吴超高原毛旭虎郭刚童文德郭鹰刘开云
Owner ARMY MEDICAL UNIV
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