Novel vaccine used for preventing tuberculosis

Abstract

The invention relates to the construction of a human GM-CSF gene and a Mycobacterium tuberculosis CFP10 gene chimerically expressed GMCSF-CPF10 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 CFP10 gene are inserted into the sequence of the same escherichia coli-Mycobacterium tuberculosis shuttle plasmid pMV361 by gene engineering technology so as to construct a recombinant shuttle plasmid rpMV361GMCSF-CFP10; and a vector is introduced into a BCG vaccine by an electroporation method so as to construct a recombinant BCG vaccine rBCG:GMCSF-CFP10. The recombinant BCG vaccine constructed by the invention can express the human GM-CSF and Mycobacterium tuberculosis CFP10 chimeric protein GMCSF-CFP10 stably, and has an immunogenicity superior to that of the conventional traditional BCG vaccine. The invention provides a process for preparing the recombinant BCG vaccine, researches the immunity thereof, and belongs to a gene engineering field and a tuberculosis vaccine field. The novel vaccine prevents the generation and the propagation of tuberculosis more effectively.

Description

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 immunodeficiency virus (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-...

Examples

Embodiment 1

[0041] Example 1 Construction, expression and identification of hGMCSF-CFP10 chimeric gene recombinant BCG

[0042] 1 material

[0043] 1.1 Strains and plasmids

[0044] 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.

[0045] 1.2 Main reagents

[0046] prime STAR TM HS DNA polymerase, dNTP, and DNA ligation kits are products of Takara; EcoR I, HindIII, and protein molecular weight standards were purchased from Jingmei; Omega plasmid extraction kits, PCR product recovery kits, gel recovery kits, and bacterial genome extraction reagents The box was purchased from Baoxin Biology; CFP10 monoclonal antibody was a product of Abcam; HRP enzyme-labeled goat anti-mouse IgG and ECL color development kit were purchased from Beijing Zhongshan Company.

[0047] 1.3 Main instruments

[0048] The PCR instrument and electrotransfer instrum...

Embodiment 2

[0073] Example 2 Study on Immunogenicity of Recombinant BCG rBCG:GMCSF-CFP10

[0074] 1 material

[0075] 1.1 Experimental animals

[0076] BALB / C mice were purchased from the Experimental Animal Center of West China Campus, Sichuan University.

[0077] 1.2 Main reagents

[0078] 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.

[0079] 1.3 Main instruments

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

[0081] 2 methods

[0082] 2.1 Animal immunity

[0083] 32 6-week-old BALB / C mice (male or female), 8 in each group, were randomly divided into PBST group, BCG group, rBCG:361 group and rBCG:GMCSF-CFP10 group, and each group was fed adaptively for 1 day. One week later, PBST (containing 0.05% Tween80), BCG and rBCG (5×10 6 CFU / only, diluted to 0.1mL with...

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 immunodeficiency virus (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-...

Examples

Embodiment 1

[0041] Example 1 Construction, expression and identification of hGMCSF-CFP10 chimeric gene recombinant BCG

[0042] 1 material

[0043] 1.1 Strains and plasmids

[0044] 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.

[0045] 1.2 Main reagents

[0046] prime STAR TM HS DNA polymerase, dNTP, and DNA ligation kits are products of Takara; EcoR I, HindIII, and protein molecular weight standards were purchased from Jingmei; Omega plasmid extraction kits, PCR product recovery kits, gel recovery kits, and bacterial genome extraction reagents The box was purchased from Baoxin Biology; CFP10 monoclonal antibody was a product of Abcam; HRP enzyme-labeled goat anti-mouse IgG and ECL color development kit were purchased from Beijing Zhongshan Company.

[0047] 1.3 Main instruments

[0048] The PCR instrument and electrotransfer instrum...

Embodiment 2

[0073] Example 2 Study on Immunogenicity of Recombinant BCG rBCG:GMCSF-CFP10

[0074] 1 material

[0075] 1.1 Experimental animals

[0076] BALB / C mice were purchased from the Experimental Animal Center of West China Campus, Sichuan University.

[0077] 1.2 Main reagents

[0078] 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.

[0079] 1.3 Main instruments

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

[0081] 2 methods

[0082] 2.1 Animal immunity

[0083] 32 6-week-old BALB / C mice (male or female), 8 in each group, were randomly divided into PBST group, BCG group, rBCG:361 group and rBCG:GMCSF-CFP10 group, and each group was fed adaptively for 1 day. One week later, PBST (containing 0.05% Tween80), BCG and rBCG (5×10 6 CFU / only, diluted to 0.1mL with...

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