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Immunologic adjuvant-Helicobacter pylori antigen fused protein oral vaccine and preparation method thereof

An immunoadjuvant and protein technology, applied in the field of biomedicine, can solve the problems of high treatment costs, extensive drug resistance, and large side effects, and achieve the effects of promoting oral absorption, solving bottlenecks and alleviating pain.

Active Publication Date: 2012-07-25
贵州贵安精准医学股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing methods for the treatment of Helicobacter pylori are mainly combined application of antibiotics and acid suppressants, but there are disadvantages such as extensive drug resistance, large side effects, high treatment costs and easy recurrence.

Method used

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  • Immunologic adjuvant-Helicobacter pylori antigen fused protein oral vaccine and preparation method thereof
  • Immunologic adjuvant-Helicobacter pylori antigen fused protein oral vaccine and preparation method thereof
  • Immunologic adjuvant-Helicobacter pylori antigen fused protein oral vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: CTB-Linker- ure B Construction of fusion gene (as shown in SEQ ID NO: 1)

[0039] Firstly, the oral immune adjuvant CTB gene and Helicobacter pylori ure B In order to ensure that the space structure of the two protein subunits is not affected after the fusion gene is expressed, the CTB gene and ure B connected in between

[0040] A connecting peptide sequence (Linker) (as shown in SEQ ID NO: 6):

[0041] AGATCCGCCGCCACCAGATCCACCACCGCCGGATCCACCGCCACC encodes a flexible peptide segment of (Gly4Ser)3, ensuring that the spatial structure of the two protein subunits in the fusion protein is not affected. This connecting peptide was originally designed by Husotn et al. in 1988 based on the X-ray diffraction analysis data of Fab fragments. Later, it was found that the peptide was longer and softer, which could reduce the steric hindrance between each protein subunit in the fusion protein, thereby making it more It is beneficial to the correct folding of each dom...

Embodiment 2

[0057] Example 2: Recombinant transfer plasmid pBacPAK8-(CTB-Linker- ure B ) construction

[0058] To embodiment 1 gained CTB-Linker- ure B The 5' and 3' ends of the fusion gene were double-digested with BamH I enzyme and EcoR I enzyme (Fermentas company), respectively, and the digested fragment was recovered by agarose gel electrophoresis; at the same time, the pBacPAK8 vector was digested with BamH I enzyme and EcoR I enzyme (Invitrogen Company) for enzyme digestion and use agarose gel electrophoresis to recover the digested fragment; then use T4 ligase (Fermentas Company) to connect the recovered fusion gene to the digested pBacPAK8 vector, so that the recombinant gene is placed in the polyhedrin Under the control of the gene promoter, transforming TG 1 Escherichia coli competent cells (Invitrogen Company), positive clones were screened to obtain the recombinant transfer plasmid pBacPAK8-(CTB-Linker- ure B ) (See Figure 5 ), it was subjected to gene sequencing, and it ...

Embodiment 3

[0059] Embodiment 3: Bombyx mori recombinant baculovirus BmNPV-(CTB-Linker- ure B ) construction

[0060] 1. Obtaining Linearized Viral DNA

[0061] BmN cells (Invitrogen) were inoculated with silkworm baculovirus Bm-BacPAK6 (Clontech). After culturing at 27°C for 72 h, the virus culture medium was collected, and the genomic DNA of Bm-BacPAK6 virus was extracted, and used Bsu36 The endonuclease digests the Bm-BacPAK6 viral genome to linearize it.

[0062] 2. Co-transfection of recombinant transfer vector plasmid and linearized viral DNA

[0063] (1) Place well-growing BmN cells in a 50 ml culture flask and culture at 27°C for 4-12 hours to allow the cells to adhere to the wall;

[0064] (2) Take A centrifuge tube and add the recombinant transfer vector plasmid pBacPAK8-(CTB-Linker- ure B ) 5 μl, 20 μl (about 1 μg) of linearized viral DNA, and make up the total volume with HBS (20mM HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; 15mM NaCl) to 50 μl, mix wel...

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Abstract

The invention relates to an immunologic adjuvant-Helicobacter pylori antigen fused protein oral vaccine and a preparation method and product thereof, belonging to the technical field of biomedicine. A connecting peptide sequence section is connected with the downstream part of an oral immunologic adjuvant gene by using a PCR (Polymerase Chain Reaction) technology; the oral immunologic adjuvant gene connected with the connecting peptide sequence is fused with a protein antigen gene by using the overlap extension RCR technology; and then, a recombination silkworm baculovirus of the fused gene is established and a target protein is expressed by using a silkworm bioreactor. According to the fused protein oral vaccine provided by the invention, the immunologic adjuvant-Helicobacter pylori antigen fused protein is expressed by using the silkworm bioreactor, so that the protein is effectively expressed, and the protein fusion expression of the immunologic adjuvant and the Helicobacter pyloriantigen is used as an intra-molecular immunologic adjuvant to enhance the immune effects.

Description

technical field [0001] The present invention relates to an oral vaccine of immune adjuvant and Helicobacter pylori antigen fusion protein and its preparation method, in particular to the preparation of recombinant plasmid and recombinant virus in the preparation of immune adjuvant and Helicobacter pylori antigen fusion protein oral vaccine, which belongs to biomedicine technology field. Background technique [0002] Helicobacter pylori (Hp), first discovered by Barry J. Marshall and J. Robin Warren in 1983, is a Gram-negative bacterium with unipolar, multi-flagellated, blunt-rounded ends, and spiral bends. A major risk factor for active gastritis, duodenal ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. In 1994, the World Health Organization / International Agency for Research on Cancer (WHO / IARC) designated Helicobacter pylori as a class I carcinogen. At present, nearly 50% of the world's population is infected with Helicobacter pylori,...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/66C07K19/00A61K39/02A61P31/04
Inventor 高国舒特俊陈剑清张耀洲
Owner 贵州贵安精准医学股份有限公司
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