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Construction method and application of recombinant drug-resistant BCG strain

A construction method and technology of drug-resistant bacteria, applied in the field of genetic engineering, can solve problems such as inability to cause immunity and microecological protection, and achieve the effects of reducing the risk of re-infection, accelerating death, and protecting patients

Active Publication Date: 2020-07-31
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Their common disadvantage is that after the end of treatment, they cannot cause sustained immune and microecological protection.

Method used

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  • Construction method and application of recombinant drug-resistant BCG strain
  • Construction method and application of recombinant drug-resistant BCG strain
  • Construction method and application of recombinant drug-resistant BCG strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The preparation method of embodiment 1 drug-resistant BCG bacterial strain

[0027] 1. Preparation of 7H9 and 7H11 medium

[0028] 1. 7H9 liquid medium: 4.7g 7H9 powder, 2mL glycerol, 900mL ddH 2 O, high temperature and high pressure sterilization, add 100mL OADC to use after cooling;

[0029] 2. 7H11 solid medium: 19g 7H11 powder, 5mL glycerol, 900mL ddH 2 O, high temperature and high pressure sterilization, add 100mL OADC to use after cooling;

[0030] 2. Screening for spontaneous drug-resistant BCG

[0031] 1. According to the MIC of M.bovis BCG (hereinafter referred to as wild-type BCG bacteria, provided by Johns Hopkins University School of Medicine) to streptomycin is 0.25 μg / mL, prepare 7H11 plate containing streptomycin (final concentration is 0.25* MIC-8*MIC) and 7H11 blank board. After diluting the M.bovis BCG grown to the logarithmic phase to an appropriate concentration, pipette 500 μL of the bacterial solution and spread it evenly on the plate. Place ...

Embodiment 2

[0044] Example 2 Construction of recombinant drug-resistant BCG strains

[0045] 1. Preparation of recombinant plasmids containing Ag85b gene and Rv2628 gene fragments.

[0046] 1. PCR product recovery and digestion

[0047] The plasmid construction process is as follows figure 1 . Using the genomic DNA of Mtb H37Rv (provided by Guangzhou Chest Hospital) as a template, the Ag85b gene was amplified by PCR (primer pair——F:

[0048] 5'-CGGGATCCATGAGACGACTTTGACGCCCGAA-3', R: 5'- CCCCTGCAGGGATCCTTAGACCGCAACGGCAATCT-3') and Rv2628 gene (primer pair - F:

[0049] 5'-GGAATTCCATATGATGTCCACGCAACGACCGA-3', R: 5'-CCCCTGCAGGGATCCTTAGACCGCAACGGCAATCT-3') fragment, the PCR product was recovered after 0.8% agarose gel electrophoresis and identified by sequencing.

[0050] The recovered Rv2628 fragment after identification and the starting plasmid p60LuxN (constructed by our laboratory, see the figure for the plasmid map, and refer to the related content of CN 201810359440.4 for the specif...

Embodiment 3

[0065] Plasmid stability detection of embodiment 3 recombinant drug-resistant BCG

[0066] The obtained recombinant drug-resistant BCG bacterial solution was diluted to an appropriate concentration, cultured in 7H9 medium containing hygromycin, and continuously subcultured five times. The bacterial solutions passed down to the fourth and fifth generations were respectively serially diluted, and the bacterial solutions were spread on 7H11 plates containing hygromycin, and cultured in an incubator for four weeks. Pick a single colony for amplification and culture and extract its DNA, use primers to amplify and identify Mark-F (5'-CGATGTGGTCGGATAGGCA-3') and Mark-R (5'-ACTCACCTGCGGTTTATCTGC-3'), and amplify 0.6 The kb fragment includes the Ag85B gene and the Rv2628 gene.

[0067] Randomly pick colonies from 5 consecutive subcultures, and use the above primer pair (Mark-F and Mark-R) to carry out colony PCR. The results are as follows: figure 2 ;Depend on figure 2 It can be o...

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Abstract

The invention discloses a construction method and application of a recombinant drug-resistant BCG strain, M.bovis BCG is used as an original bacterium to construct a drug-resistant BCG strain with resistance to at least one of streptomycin, levofloxacin, ethambutol, protionamide, p-aminosalicylic acid and amikacin; and sequence fragments capable of expressing related antigens Ag85b and Rv2628 causing immune response can be further inserted to construct a recombinant drug-resistant BCG strain. The recombinant drug-resistant BCG strain can compete with Mtb in growth to accelerate death of the Mtb, and when the recombinant drug-resistant BCG strain is combined with a drug for treating tuberculosis, the treatment effect on Mtb (sensitive Mtb and drug-resistant Mtb) can be further enhanced, re-infection of a patient can be avoided, and an important medical prospect is achieved.

Description

technical field [0001] The invention belongs to the field of genetic engineering and novel vaccine development, and relates to a BCG strain, in particular to a construction method and application of a recombinant drug-resistant BCG strain. Background technique [0002] Tuberculosis, commonly known as tuberculosis and "white plague", is an ancient and long-standing infectious disease, and one of the serious diseases that have long endangered human health. It is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) infection and is the pathogenic bacteria that causes tuberculosis (TB). Mtb can invade various organs of the human body, and the lungs are the most common. About one-third of the world's population is infected with tuberculosis, and about 3 million people die of tuberculosis every year, with an average of more than 8,000 deaths per day. [0003] In recent years, due to the dual infection of HIV and Mycobacterium tuberculosis and the prevalence of...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K39/04A61P31/06C12R1/32
CPCC07K14/35C12N15/74A61K39/04A61P31/06A61K2039/52C12R2001/32C12N9/0073C12N9/1077C12N9/90A61K2039/523A61K2039/522
Inventor 张天宇王邦兴朱利叶斯·恩迪朗古·穆格文鲁奇瓦拉·李雾刘志永
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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