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A kind of recombinant mycobacterium smegmatis strain of knocking out c-di-amp decomposing enzyme and its application

A technology of Mycobacterium smegmatis and Mycobacterium strains, applied in the field of tuberculosis vaccine and immunotherapy

Active Publication Date: 2020-10-09
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still no recombinant vaccine available for prevention and treatment, indicating that extensive screening of new Mtb antigens is still needed to obtain recombinant mycobacteria with better immune effects for the prevention and treatment of TB

Method used

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  • A kind of recombinant mycobacterium smegmatis strain of knocking out c-di-amp decomposing enzyme and its application
  • A kind of recombinant mycobacterium smegmatis strain of knocking out c-di-amp decomposing enzyme and its application
  • A kind of recombinant mycobacterium smegmatis strain of knocking out c-di-amp decomposing enzyme and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0042] Construction of MSMEG_2630 Gene Homologous Recombination Knockout Plasmid

[0043] According to Ms MC 2 Design primers for the upstream and downstream sequences of the genome MSMEG_2630 gene of 155 strains:

[0044]

[0045] Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. Take Ms MC 2 The genome of 155 strains was used as a template, and the target gene was amplified by PCR. The reaction parameters were: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles, and extension at 72°C for 5 minutes after the last cycle. 1% agarose gel electrophoresis analysis showed that 456bp and 468bp target bands were amplified respectively. The target fragments were recovered by the gel extraction kit, digested separately, ligated with the pMSG360 plasmid recovered by the same digestion, and transformed into E.coli DH5α competent cells, and screened with bleomyci...

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Abstract

The invention relates to a recombinant mycobacterium smegmatis strain with a knockout c-di-AMP clastic enzyme. Upstream and downstream homologous arms of a mycobacterium smegmatis c-di-AMP degrading enzyme gene MSMEG-2630 are connected to a pMSG360 carrier separately; recombinant plasmids are transformed into a mycobacterium smegmatis competent cell containing recombinase gp60 and gp61pJV53 plasmid, and the recombinant mycobacterium smegmatis strain (rMS-deltaCnpB) with the knockout c-di-AMP clastic enzyme is screened and obtained, wherein the preservation number is CCTCC M 2016336. Mice are immunized by the strain rMS-deltaCnpB, mice humoral and cell immune responses, especially a mycobacterium tuberculosis-infected Th1 cell immune response can be adjusted, and the protective ability of the organism resistant to mycobacterium infection is improved, so that the recombinant mycobacterium smegmatis strain can be applied to development of vaccines and preparations for preventing and / or treating tuberculosis and has a relatively good application prospect.

Description

technical field [0001] The invention belongs to the field of tuberculosis vaccine and immunotherapy. The invention relates to a recombinant mycobacterium smegmatis strain knocking out c-di-AMP decomposing enzyme. The present invention also relates to the application of the above-mentioned recombinant Mycobacterium smegmatis strain in vaccines and preparations for preventing and / or treating tuberculosis. Background technique [0002] Tuberculosis Epidemic Status [0003] Tuberculosis (TB) is a chronic infectious disease that seriously endangers human health worldwide. Mycobacterium tuberculosis (Mtb) is the causative agent of TB. According to the WHO report, in 2015, there were an estimated 10.4 million new cases worldwide, among which 1.2 million (11%) were HIV patients, and a total of 1.4 million people died in TB. TB remains in the top ten causes of death. Six countries—India, Indonesia, China, Nigeria, Pakistan and South Africa—accounted for 60% of the total number o...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K35/74A61P31/06C12R1/34
CPCA61K35/74C12N9/16
Inventor 柏银兰徐志凯王立飞宁唤唤许艳慧康健路延之周洁薛莹陆翮高子招
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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