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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, introduction of foreign genetic material, DNA/RNA fragments using vectors, etc., can solve problems such as safety doubts, and achieve the effect of excellent immunogenicity
Inactive Publication Date: 2010-09-08
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 GMCSF-CFP10-ESAT6 chimeric gene recombinant BCG
[0046] 1 material
[0047] 1.1 Strains and plasmids
[0048] The BCG Shanghai strain was provided by the Chengdu Institute of Biological Products, the plasmid pMV361 was preserved in our laboratory, and the pORF-hGMCSF plasmid was purchased from Invitrogen.
[0049] 1.2 Main reagents
[0050] prime STAR TM HS DNApolymerase, dNTP, and DNAligation kits are products of Takara; EcoR I, HindIII, and protein molecular weight standards were purchased from Jingmei; Omegaplasmid extraction kits, PCR product purification kits, gel recovery kits, and bacterial genome extraction reagents The box was purchased from Baoxin Biology; the human GM-CSF monoclonalantibody was a product of Abcam; the HRP enzyme-labeled goat anti-mouse IgG and ECL color development kit were purchased from Beijing Zhongshan Company.
[0084] 1.1 Experimental animals BALB / C mice were purchased from the Experimental Animal Center of West China Campus, Sichuan University.
[0085] 1.2 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 antibody was purchased from eBioscience, RPMI1640 medium and fetal bovine serum were purchased from Gibco.
[0089] Take 32 6-week-old BALB / C mice (male or female), 8 in each group, and randomly divide them into PBST group, BCG group, rBCG:361 group and rBCG:GMCSF-CFP10-ESAT6 group. After feeding for 1 week, PBST (0.05% Tween80), BCG and rBCG (5×10 6 CFU / only, diluted to 0.1mL with PBST), immunized once in total. After immunization, ob...
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Technical field: [0001] The invention relates to the fields of genetic engineering and novel 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 floating population, the co-infection of human immunodeficiencyvirus (HIV) and Mycobacterium tuberculosis, and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis , which increases the global tuberculosis epidemic situation and poses new challenges to the global tuberculosis prevention and control. BCG (Bacille Calmette Guerin, BCG) is currently the only vaccine used to prevent tuberculosis, but its immune protection effect is extremely unstable, and the immune protection effect of the population in different regions varies greatly after being inoculated with BCG (the protection rate ranges from 0-80%) ). The development of a more effective anti...
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