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Improved recombinant bacillus calmetter Guerin (BCG)

A technology of BCG and gene recombination, which is applied in the direction of recombinant DNA technology, DNA/RNA fragments, and the use of vectors to introduce foreign genetic materials, etc. It can solve the problems of insufficient immune protection, low vector expression efficiency, and insufficient antigen stimulation, etc., to achieve safety and efficiency The effect of the immune effect

Inactive Publication Date: 2010-06-09
SICHUAN UNIV
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AI Technical Summary

Problems solved by technology

Using ESAT6 or Ag85A recombinant BCG alone, after immunizing animals, it is found that its immune protection is not as good as that of BCG. The reason may be that its antigen stimulation is insufficient, and on the other hand, the expression efficiency of its vector may be low.

Method used

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  • Improved recombinant bacillus calmetter Guerin (BCG)
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  • Improved recombinant bacillus calmetter Guerin (BCG)

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Experimental program
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Embodiment Construction

[0027] 1. Design and synthesis of primers

[0028] Design the following 2 pairs of primers according to the gene sequence reported in Genbank: P1: 5'-GC C AAT TG T AAT GCA GCTTGT TGA CAG GGT T-3'; P2:5'- GCT TCC ACC TCC TCC GCT TCC ACC ACC TCC GCT TCC ACC GCC ACC GGC GCC CTG GGG CGC GGG-3'; P3:5'- GGT GGC GGT GGA AGC GGA GGT GGT GGA AGC GGA GGA GGT GGA AGC ACA GAG CAG CAG TGG AAT-3'; P4:5'-GC T TCG AA T CTA TGC GAA CAT CCC AGT GAC-3'. The underlined parts in P1 and P4 are Mun I and NspV restriction sites respectively, and the underlined parts in P2 and P3 are complementary 45 base linkers. Synthesized and purified by Shanghai Invitrogen Company.

[0029] 2. PCR amplification of the target gene

[0030] PCR amplification conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 62°C renaturation for 1 min, 72°C extension for 1 min, a total of 30 cycles, and finally 72°C extension for 10 min. Using H37RV genomic DNA as a template, first use P1...

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Abstract

The invention discloses an improved recombinant bacillus calmetter Guerin (BCG), belonging to the technical field of new medicaments. The immune protecting effect of a unique anti-tuberculosis vaccine BCG for tuberculosis is not exact and the tuberculosis morbidity gradually rises over the past 10 years. Therefore, the development of a more effective anti-tuberculosis vaccine is important. Gene sequences of mycobacterium tuberculosis ESAT6 and Ag85A are inserted into shuttle plasmids of colon bacillus-mycobacterium tuberculosis for forming recombinant plasmids; and the recombinant plasmids are converted into the BCG to form a recombinant anti-tuberculosis vaccine. Proved by research, the recombinant BCG is used for expressing introduced foreign genes and is a novel anti-tuberculosis vaccine.

Description

technical field [0001] The invention belongs to the field of biology and new medical technology (medical biotechnology). Background technique [0002] BCG (bacillus calmette querin, BCG) is the only vaccine to prevent tuberculosis, but its immune protection effect is unstable, and its preventive effect on adult tuberculosis is 0-80%. Scholars from various countries are working on the research of more efficient and safer tuberculosis vaccines. Introducing exogenous genes into BCG and making exogenous DNA continuously and stably expressed in BCG, that is, recombinant BCG, is currently a hot spot in the development of new tuberculosis vaccines. Whether the effect of recombinant BCG is ideal or not depends on the nature of the foreign gene introduced into it and the expression efficiency of the foreign DNA. A large number of studies have shown that Mycobacterium tuberculosis immunoprotective antigen Ag85A and early secretory antigen target 6 (Early secretory antigen target 6, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/04C12N15/62C12N15/74A61P31/06
Inventor 鲍朗邓仪昊杨晓玲
Owner SICHUAN UNIV
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