Recombinant BCG vaccine for tuberculosis prevention
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A technology of recombinant BCG and BCG, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, DNA / RNA fragments, etc., can solve problems such as safety concerns, and achieve excellent immunogenicity
Inactive Publication Date: 2013-07-31
SICHUAN UNIV
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Although the first three methods can stimulate the body's protective immune response to varying degrees, their safety has also been questioned
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Embodiment 1
[0045] Example 1 Construction, expression and identification of recombinant BCG vaccine of GMCSF-CFP10-ESAT6 chimeric gene
[0046] 1 material
[0047] 1.1 Strains and plasmids
[0048] 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.
[0049] 1.2 Main reagents
[0050] prime STAR TM HS DNA polymerase, dNTP and DNA ligation kits are products of Takara; EcoRI, HindIII and protein molecular weight standards were purchased from Jingmei; Omega plasmid extraction kit, PCR product purification kit and gel recovery kit, bacterial genome extraction reagent The box 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 reagent kit was purchased from Beijing Zhongshan Company.
[0051] 1.3 Main instruments
[0052] The PCR machine and the electric transfer machine we...
Embodiment 2
[0082] Example 2 Recombinant BCG vaccine rBCG: GMCSF-CFP10-ESAT6 immunogenicity study
[0083] 1 material
[0084] 1.1 Experimental animals BALB / C mice were purchased from the Experimental Animal Center of West China Campus of Sichuan University.
[0085] 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.
[0089] Take 32 BALB / C mice aged 6 weeks (male or female), 8 mice in each group, randomly divided into PBST group, BCG group, rBCG:361 group and rBCG:GMCSF-CFP10-ESAT6 group, each group is adaptable After feeding for 1 week, PBST (0.05% Tween80), BCG and rBCG (5×10 6 CFU per mouse, diluted with PBST to 0.1 mL), immunized once. Afte...
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Abstract
The invention relates to construction of a BCG vaccine through chimeric expression of GMCSF-CFP10-ESAT6 protein with a human GM-CSF gene, and a CFP10 gene and an ESAT6 gene of mycobacterium tuberculosis and research on immunogenicity of the BCG vaccine. In the construction method, a human GM-CSF gene sequence and a CFP10 gene sequence and an ESAT6 gene sequence of the mycobacterium tuberculosis are inserted into one escherichia coli-mycobacterium tuberculosis shuttle plasmid pMV361 sequence through the genetic engineering technology to construct a recombinant shuttle plasmid rpMV361GMCSF-CFP10-ESAT6. Then the carrier is guided into a BCG vaccine through electroporation to construct a recombinant BCG vaccine rBCG: GMCSF-CFP10-ESAT6. The recombinant BCG vaccine constructed in the invention can stably express the GMCSF-CFP10-ESAT6 chimeric protein, and the immunogenicity of the recombinant BCG vaccine is superior to that of the immunogenicity of the traditional BCG vaccine. The inventionalso provides a preparation process of the recombinant BCG vaccine and research on the immunity of the recombinant BCG vaccine, which belong to the genetic engineering field and the tuberculosis vaccine field. Moreover, the invention can prevent the occurrence and spread of the 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 a more effective anti-tuber...
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