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Novel recombinant vaccine used for preventing tuberculosis

A technology of recombinant BCG and recombinant plasmid, which is applied in the direction of recombinant DNA technology, application, medical preparations containing active ingredients, etc., can solve the problem of low immune protection effect and achieve the effect of excellent immunogenicity

Inactive Publication Date: 2010-10-06
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bao constructed two rBCG strains expressing Hsp60-ESAT6 fusion protein and ESAT6 secreted protein respectively, the former induced higher titers of specific antibodies and stronger cellular immune responses than BCG, while the latter had similar immunogenicity to the parental BCG strain , the virulence of the two strains of rBCG has not been enhanced, and both can obviously resist MTB infection, but the immune protection effect is still lower than that of BCG

Method used

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  • Novel recombinant vaccine used for preventing tuberculosis
  • Novel recombinant vaccine used for preventing tuberculosis
  • Novel recombinant vaccine used for preventing tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction, expression and identification of recombinant BCG vaccine of hGMCSF-ESAT6 chimeric gene

[0045] 1 material

[0046] 1.1 Strains and plasmids

[0047] The BCG Shanghai strain was provided by Chengdu Institute of Biological Products, the plasmid pMV361 was kept in our laboratory, and the pORF-hGMCSF plasmid was purchased from Invitrogen.

[0048] 1.2 Main reagents

[0049] prime STAR TM HS DNA polymerase, dNTP, and DNA ligation kit are products of Takara; EcoRI, HindIII and protein molecular weight standards were purchased from Jingmei; Omega plasmid extraction kit, PCR product recovery kit and gel recovery kit, bacterial genome extraction reagent The kit was purchased from Baoxin Bio; the human GM-CSF monoclonal antibody was a product of Abcam; the HRP enzyme-labeled goat anti-mouse IgG and ECL color development kit was purchased from Beijing Zhongshan Company.

[0050] 1.3 Main instruments

[0051] The PCR machine and the electric transfer machine were pu...

Embodiment 2

[0076] Example 2 Study on the immunogenicity of recombinant BCG vaccine rBCG:GMCSF-ESAT6

[0077] 1 material

[0078] 1.1 Experimental animals BALB / C mice were purchased from the Experimental Animal Center of West China Campus of Sichuan University.

[0079] 1.2 The main reagents IFN-γ, IL-4 ELISA detection kits were purchased from R&D company, PE-labeled CD 4 And FITC labeled CD 8 Anti-mouse primary antibodies were purchased from eBioscience, and RPMI1640 medium and fetal bovine serum were purchased from Gibco.

[0080] 1.3 Main instruments: FACSCalibur flow cytometer (BD Biosciences), microplate reader (Bio-Rad).

[0081] 2 method

[0082] 2.1 Animal immunity

[0083] Take 32 6-week-old BALB / C mice (male or female), 8 mice in each group, randomly divided into PBST group, BCG group, rBCG361 group and rBCG:GMCSF-ESAT6 group, each group is adaptively reared for 1 week , Inject PBST (0.05% Tween80), BCG and rBCG (5×10 6 CFU per mouse, diluted with PBST to 0.1 mL), immunized once. After im...

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Abstract

The invention relates to the construction of a human GM-CSF gene and Mycobacterium tuberculosis ESAT6 gene chimerically expressed GMCSF-ESAT6 protein recombinant Bacillus Calmette Guerin (BCG) vaccine and immunogenicity research thereof, namely the sequence of the human GM-CFS gene and the sequence of the Mycobacterium tuberculosis ESAT6 gene are inserted into the sequence of the same escherichiacoli-Mycobacterium tuberculosis shuttle plasmid pMV361 by gene engineering technology so as to construct a recombinant shuttle plasmid rpMV361GMCSF-ESAT6; and a vector is introduced into a BCG vaccine by an electroporation method so as to construct a recombinant BCG vaccine rBCG:GMCSF-ESAT6. The recombinant BCG vaccine can express the human GM-CSF and Mycobacterium tuberculosis ESAT6 gene chimeric protein GMCSF-ESAT6 stably, and has an immunogenicity superior to that of the conventional BCG vaccine. The invention provides a process for preparing the recombinant BCG vaccine, researches the immunity thereof, and belongs to the field of gene engineering and the field of tuberculosis vaccine. The novel recombinant vaccine prevents the generation and the propagration of tuberculosis more effectively.

Description

Technical field: [0001] The invention relates to the field of genetic engineering and the field of new tuberculosis vaccines, in particular to a recombinant BCG vaccine. Background technique: [0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. In recent years, with the increase of the floating population, the concurrent infection of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis, and the emergence of multi-drug resistant strains of Mycobacterium tuberculosis , To increase the global tuberculosis epidemic situation, and present new challenges to global tuberculosis prevention and treatment. Bacille Calmette Guerin (BCG) is currently the only vaccine used to prevent tuberculosis, but its immune protection effect is extremely unstable. The immune protection effect of people in different regions after being vaccinated with BCG varies greatly (protection rate ranges from 0-80%) ). The development of an anti-tuberculosis vaccin...

Claims

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Application Information

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IPC IPC(8): A61K39/04A61K39/39A61K48/00A61P31/06C12N15/31C12N15/63
Inventor 鲍朗杨晓玲邓仪昊
Owner SICHUAN UNIV
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