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Construction and application of PhoPR gene overexpressed BCG strain

A technology of gene overexpression and BCG, applied in the field of vaccine research, can solve the problems of unstable protective effect, BCG can not meet the human tuberculosis prevention, complications or disseminated tuberculosis and other problems

Pending Publication Date: 2019-08-02
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cultivation of BCG is simple, the price is low, and its safety is relatively good, but people have gradually discovered its shortcomings and deficiencies in the long-term application process: 1. WHO believes that BCG vaccination is effective for children with severe tuberculosis such as tuberculous meningitis and miliary pulmonary tuberculosis It has a good preventive effect, but it has no protective effect on adult tuberculosis, but adults are the main source of tuberculosis transmission, so BCG vaccination cannot reduce tuberculosis transmission
2. The protective effect is not stable, and there are relative application contraindications and adverse reactions
3. For children with congenital immunocompromise and patients with acquired immunodeficiency, BCG vaccination may cause serious complications or disseminated tuberculosis; 4. Some important antigen genes are lost during the long-term passage, and immune protection The effectiveness is reduced, and the BCG vaccination will also have a certain impact on the diagnostic results of tuberculosis patients
[0004] The traditional BCG vaccine can no longer meet the needs of human tuberculosis prevention, and researchers are constantly working hard to develop new tuberculosis vaccines
Many emerging tuberculosis vaccines are in different stages of testing, and so far there is no vaccine that can successfully replace BCG and is widely used in clinical prevention

Method used

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  • Construction and application of PhoPR gene overexpressed BCG strain
  • Construction and application of PhoPR gene overexpressed BCG strain
  • Construction and application of PhoPR gene overexpressed BCG strain

Examples

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Effect test

Embodiment 1

[0024] Example 1 Construction and Identification of Overexpressed PhoPR Gene Recombinant Plasmids

[0025] 1. Materials and methods: 1.1 Main reagent materials: Main materials: Bacillus Calmette-Guerin strain (BCG strain for short) was provided by the Institute for the Control of Biological Products of Traditional Chinese Medicine and kept in our laboratory; restriction endonucleases Mun I, Hind III, and Pvu II were obtained from Canada Fermentas Biological Company; T4 DNA ligase, Prime Script RT Master Mix from Dalian Bao Biological Engineering Co., Ltd.; plasmid mini-extraction kit (spin column type), agarose gel recovery kit (spin column type), DNAMarker, 2×Taq PCR MasterMix comes from Beijing Tiangen Biochemical Technology Co., Ltd.; OXOID tryptone, OXOID yeast extract British Tryptone Company. β-mercaptoethanol was from Shanghai Sangon Bioengineering Technology Co., Ltd.; Tris base (Tris, base) and lysozyme were from Amresco; FDA was from Sigma; Rhodamine B was from Solar...

Embodiment 2

[0037] Construction and Identification of Example 2 Bacillus Calmette-Guerin PhoPR Gene Overexpression Strain

[0038] 2.1 Competent preparation of BCG strains. (1) The BCG strain was inoculated into Middlebrook 7H9 medium supplemented with serum albumin, 0.5% Tween 80, 0.5% glycerol and 0.3% catalase, and cultured in a shaker at 200rpm / min at 37°C until the logarithmic growth phase ( OD600 reaches about 0.5). Collect the bacterial solution with a pre-cooled 50mL centrifuge tube, and centrifuge at 4,000rpm at 4°C for 10 minutes after ice bathing for 1.5h; discard the supernatant, add 20mL of 10% pre-cooled glycerin to wash the precipitate, and centrifuge again at 4°C at 4,000rpm Centrifuge for 10 min; repeat washing once. Add 10% pre-cooled glycerol to resuspend the pellet, and store at -20°C for electroporation; (2) Electrotransformation of recombinant plasmids and construction and identification of overexpressed mutant BCG in the PhoPR two-component system. Mix 10 μl of r...

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Abstract

The invention relates to a construction and an application of a novel PhoPR gene overexpressed BCG (Bacillus Calmette-Guerin) mutant strain. The construction comprises the following steps: firstly, converting a carrier containing a BCG overexpressed PhoPR gene into BCG, screening, detecting and confirming a successfully converted strain as the novel PhoPR gene overexpressed BCG mutant strain. Theinvention relates to the novel PhoPR gene overexpressed BCG mutant strain, namely, BCG: PhoPR strain. Such a strain is mainly used for researching the functions of BCG PhoPR gene overexpressed associated protein PhoPR at the aspects of growth of mycobacterium tuberculosis in host, drug tolerance, permeability regulation, acid resistance, pathogenicity, and the like, and is used for developing andresearching novel vaccines for interfering and treating tuberculosis.

Description

technical field [0001] The invention belongs to recombinant genes, and relates to the construction and application of a BCG PhoPR gene overexpression strain. The invention relates to the field of vaccine research, constructing a BCG PhoPR gene overexpression strain, which is used for the research potential and vaccine development of the overexpression of the BCG PhoPR two-component system-related protein PhoP / R on the virulence intensity and immune protection of new BCG mutant strains, etc. . Background technique [0002] Tuberculosis is a worldwide infectious disease caused by Mycobacterium Tuberculosis infection, and it is also the infectious disease with the highest mortality rate caused by a single bacterial infection. Tuberculosis is still the number one killer of infectious diseases in the world. New data released by the World Health Organization on September 18, 2018 in the "Global Tuberculosis Control Report 2018" showed that in 2017, an estimated 10 million people...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12R1/32
CPCC07K14/35C12N15/74
Inventor 张万江王洪洲吴芳吴江东张杰董江涛柳小玲朱荟云赵正涌赵彦恒邵萌
Owner SHIHEZI UNIVERSITY
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