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Recombinant fusion protein vaccine and attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection

A technology of Helicobacter pylori and fusion protein, which is applied in the field of recombinant fusion protein vaccine and attenuated live bacteria vector vaccine, and can solve problems such as easy generation of tolerance

Inactive Publication Date: 2010-12-08
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In summary, fully and comprehensively utilizing the antigenic properties of Hp CagA, VacA and UreB, so that these antigenic properties can be brought into play synergistically, and overcome the problems of easy tolerability in mucosal immunity, and provide a method for Vaccines for the treatment and prevention of Hp infection will bring great benefits to patients with gastritis and peptic ulcer, but there are no reports in this regard at present

Method used

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  • Recombinant fusion protein vaccine and attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection
  • Recombinant fusion protein vaccine and attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection
  • Recombinant fusion protein vaccine and attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Obtaining HpDNA fragment cagA 160 , VacA 62 And ureB 138

[0069] 1. Primer design and synthesis

[0070] Design primers based on the nucleic acid sequence of the Hp 26695 genome (NC_000915) published by GenBank. The 5'end of the upstream primer introduces the EcoR I restriction site, the 5'end of the downstream primer introduces the Hind III restriction site, and the middle primer introduces the flexible Linker (GGGSGGGS) sequence( Picture 9 ).

[0071] The primer design is as follows:

[0072]

[0073] The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0074] 2. PCR amplification of target DNA

[0075] 1) Preparation of template

[0076] Use "Bacterial Genomic DNA Extraction Kit" (TIANGEN, DP302) to extract the genome of Hp 26695 (purchased from American Type Culture Collection, ATCC, preserved by this unit, and can be released to the public as a verification test) genome, The operation is carried out according to the instructions.

[0077] 2...

Embodiment 2

[0081] Example 2 Construction of cagA 160 -vacA 62 -ureB 138 Fusion gene

[0082] 1. Obtain cagA by overlap extension PCR 160 -vacA 62 Fusion gene

[0083] Taking recycled cagA 160 And vacA 62 Gene fragment as template, cagA 160 P1 and vacA 62 P2 is the primer for PCR amplification, the reaction system is as follows:

[0084]

[0085] PCR reaction conditions are: 94°C pre-denaturation for 5min; 94°C denaturation for 30s, 56°C annealing for 30s, 72°C extension for 1min, 10 cycles; adding cagA 160 P1 (10μM) and vacA 62 P2 (10μM) 2μl each; denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 2min, 30 cycles; full extension at 72°C for 10min. After the reaction is completed, take 3μl of PCR reaction product and detect it by 2.0% agarose gel electrophoresis. figure 2 The middle shows: the size of the fusion gene fragment is consistent with the prediction, indicating that the fusion gene is obtained by overlapping extension. After agarose gel electrophoresis, re...

Embodiment 3

[0090] Example 3 Recombinant plasmid pET 22b(+)-cagA 160 -vacA 62 -ureB 138 Build

[0091] 1. Enzyme digestion

[0092] The cagA to be obtained 160 -vacA 62 -ureB 138 The fusion gene and pET 22b(+) plasmid were digested with the restriction enzymes EcoR I and Hind III of Bao Bioengineering (Dalian) Co., Ltd. respectively, and operated according to the instructions. Restriction digestion identification results such as Figure 4 Shown. Agarose gel electrophoresis, recovery of 5.5kb vector fragment and 1.1kb cagA 160 -vacA 62 -ureB 138 Fusion gene fragments.

[0093] 2. Connect

[0094] Detect the concentration of the target DNA fragment and the vector fragment by an ultraviolet spectrophotometer, and carry out the ligation reaction according to the principle that the molar ratio of the foreign fragment to the vector is generally 1:2~10 (using the ligation kit of Bao Bioengineering (Dalian) Co., Ltd. ), the connection reaction system is as follows:

[0095] cagA 160 -vacA 62 -ureB 138 E...

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Abstract

The invention relates to a recombinant fusion protein vaccine and an attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection, and belongs to the field of biopharmaceutics. The recombinant fusion protein vaccine and the attenuated live vector vaccine for expressing the recombinant fusion protein are characterized in that: the recombinant fusion protein is formed by connecting immune protective function fragments of helicobacter pylori cytotoxin relevant gene protein CagA from helicobacter pylori, vacuolization cytotoxin VacA and urease subunit UreB linearly, and the immunogenicity and immune protection of the recombinant fusion protein are verified through animal experiments. The live vaccine has the advantages that: 1, an Hp fusion protein gene can beexpressed stably; 2, mucosa and systemic immune response can be induced after immunity; and 3, the method is convenient, the cost is low, and the economic benefit is obvious. Therefore, the attenuated live vector vaccine can be used as candidate vaccines for treating and preventing the Hp infection.

Description

Technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a recombinant fusion protein vaccine for treating and preventing Helicobacter pylori infection and a live attenuated bacteria carrier vaccine. Background technique [0002] Gastritis and peptic ulcer are common and frequently-occurring diseases in humans. Gastric cancer is also one of the malignant tumors with high morbidity and mortality. Helicobacter pylori (Hp) is the main cause of chronic active gastritis and peptic ulcer. It is also closely related to the occurrence of gastric adenocarcinoma and gastric mucosa-related lymphoid tissue lymphoma (Nomura A, Stemmermann GN, Chyou PH et al. Helicobacter pylori infection and gastric carcinoma among JapaneseAmericans in Hawaii. N-Engl-J-Med. 1991, 325(16): 1132-1136.; Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N-Engl-J-Med. 1991, 325(16): 1127-1131.; Dunn BE, Cohen ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61K48/00A61P31/04C12N15/31C12N15/63
CPCY02A50/30
Inventor 邹全明刘开云吴超毛旭虎郭刚石云余抒陈立解庆华
Owner ARMY MEDICAL UNIV
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