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Reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof

A technology of recombinant BCG vaccine and chimeric gene, applied in the field of microorganisms and bioengineering, can solve the problems of weak protection, small molecular weight of antigen, etc., and achieve the effect of high-level expression

Inactive Publication Date: 2010-12-15
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ESAT-6 antigen has a small molecular weight, and although it has good immunogenicity in small animals, the immune response generated by immunizing large animals alone is very weak and has poor protection

Method used

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  • Reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof
  • Reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof
  • Reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. PCR amplification and cloning of Mycobacterium tuberculosis chimeric gene Ag856A2

[0042] Design a pair of primers whose sequences are as follows:

[0043] 85A-M1: 5′-ATA GGA TCC TTT TCC CGG CCG GGC TTG C-3',

[0044] 85A-M2: 5′-TAA GAA TTC CTA GGC GCC CTG GGG CGC-3'.

[0045] Wherein the 5' end primer (sequence 3 in the sequence listing) introduces the BamH I restriction site at the upstream of the Ag85A structural gene; the 3' end primer (sequence 4 in the sequence listing) introduces the EcoRI restriction site at the downstream of the Ag85A gene, and the primer The synthesis of was completed by Shanghai Yingjun Biotechnology Company. Using the nucleic acid vaccine HG856A plasmid DNA as a template, set the PCR reaction program as follows: 95°C×5min, (94°C×30s, 60°C×30s, 72°C×90s)×30Cycles, and finally 72°C×5min extension, obtained by PCR amplification The chimeric gene Ag85A( figure 1 , lane 3), DNA sequence see figure 2 B, and submitted to Gen...

Embodiment 2

[0046] Example 2. Construction of chimeric gene recombinant expression vector

[0047] The chimeric gene Ag856A2 fragment obtained by PCR amplification and the shuttle expression vector pMFA41 were digested by BamHI-EcoR I double enzyme digestion, separated by agarose gel electrophoresis, and the target fragment was recovered and purified, and the target gene and the carrier were mixed at a molar ratio of 3: 1 mixed and connected at 22°C for 1 hour to transform DH5α competent cells. The recombinant clone was inoculated into LB liquid medium and cultivated overnight, the recombinant plasmid was extracted, and BamHI-EcoR I double enzyme digestion verified that the chimeric gene fragment was successfully inserted into the mycobacterial expression vector pMFA41 ( figure 1 , lane 2), indicating that the chimeric gene recombinant expression plasmid was successfully constructed. For the plasmid map of the recombinant expression vector pMFA856, see figure 2 A, the amino acid sequen...

Embodiment 3

[0048] Example 3. Construction and screening of chimeric gene recombinant BCG

[0049]1) Preparation of BCG electrotransformation competent cells: lyophilized BCG strains were purchased from Shanghai Institute of Biological Products, resuspended in sterile water, streaked and activated on Middlebrook 7H11+10% OADC plates, cultured at 37°C for 3- 4 weeks until single colonies appear. Pick a single colony and inoculate it into 150mL Middlebrook 7H9+10% OADC liquid medium, culture at 37°C with slight shaking until mid-log phase (OD 600 After about 0.8), place the Erlenmeyer flask on ice for 30 minutes, cool the medium to 0°C, centrifuge at 5,000g for 5 minutes at 4°C, and recover the bacteria. Resuspend the bacterial pellet in 15 mL of ice-cold 10% oil solution, centrifuge at 5,000 g for 5 min at 4°C, and wash the cells twice. Finally, the cells were recovered by centrifugation at 5,000 g for 5 min at 4°C, resuspended in pre-cooled 10% glycerol solution with 1 / 20 volume of the ...

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Abstract

The invention provides a reinforced mosaic gene recombinant bacillus calmette-guerin, which comprises a recombinant shuttle expression vector guided into bacillus calmette-guerin (BCG) by an electro-transformation method and cloned with a bacillus tubercle mosaic gene Ag85A-ESAT6. The invention also provides a preparation method for the reinforced mosaic gene recombinant bacillus calmette-guerin and application thereof to preparation of recombinant vaccines for preventing tuberculosis. A mycobacterium differential expression system or other shuttle expression plasmid is used as the vector so as to realize high-level expression of the bacillus tubercle mosaic gene in the bacillus calmette-guerin. The reinforced mosaic gene recombinant bacillus calmette-guerin can produce reinforced TH1-type immune response aiming at Ag85A and ESAT-6 antigenic specificities respectively, and is expected to become a novel tuberculosis vaccine for replacing the bacillus calmette-guerin.

Description

technical field [0001] The invention belongs to the field of microorganisms and bioengineering. It specifically relates to a chimeric gene recombinant vaccine, and more specifically relates to a chimeric gene recombinant BCG vaccine and a preparation method thereof. Background technique [0002] Tuberculosis is a major public infectious disease of worldwide concern. As early as 1993, the World Health Organization (WHO) declared a state of emergency for the tuberculosis epidemic because of the increasing number of tuberculosis cases worldwide. At present, about 1 / 3 of the world's population (1.86 billion) carries Mycobacterium tuberculosis, with about 8 million new cases and 2 million deaths from tuberculosis every year; there are about 6 million active tuberculosis patients in my country, and there are about 6 million active tuberculosis patients in China. 250,000 people died from tuberculosis. According to the 2005 infectious disease epidemic situation announced by the Mi...

Claims

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Application Information

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IPC IPC(8): A61K39/04A61K48/00A61P31/06C12N15/31C12N15/74
Inventor 范小勇马辉赵国屏李忠明
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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