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Recombinant BCG vaccine for tuberculosis prevention

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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the first three methods can stimulate the body's protective immune response to varying degrees, their safety has also been questioned

Method used

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  • Recombinant BCG vaccine for tuberculosis prevention
  • Recombinant BCG vaccine for tuberculosis prevention
  • Recombinant BCG vaccine for tuberculosis prevention

Examples

Experimental program
Comparison scheme
Effect test

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 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 purification kits, gel recovery kits, and bacterial genome extraction reagents The box was purchased from Baoxin Biology; the human GM-CSF monoclonal antibody was a product of Abcam; the HRP enzyme-labeled goat anti-mouse IgG and ECL color development kit were purchased from Beijing Zhongshan Company.

[0051] 1.3 Main instruments

[0052] The PCR instrument and...

Embodiment 2

[0082] Example 2 Study on Immunogenicity of Recombinant BCG rBCG:GMCSF-CFP10-ESAT6

[0083] 1 material

[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.

[0086] 1.3 Main instruments FACSCalibur flow cytometer (BD Biosciences), microplate reader (Bio-Rad).

[0087] 2 methods

[0088] 2.1 Animal immunity

[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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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 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...

Claims

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Application Information

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IPC IPC(8): A61K39/04C12N15/62C12N15/74C12N1/21A61P31/06
Inventor 鲍朗杨晓玲邓仪昊
Owner SICHUAN UNIV
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