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Recombination BCG vaccine rBCG::AB

A technology of recombinant BCG and BCG, applied in the direction of recombinant DNA technology, microorganisms, bacteria, etc., to achieve a strong protective effect

Inactive Publication Date: 2010-12-22
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has been confirmed that BCG expressing Ag85B (rBCG30, University of California, USA) and BCG expressing cytolysin (Max Blank Institute of Infectious Diseases, Berlin, Germany) are more protective than BCG in immunized animals, but there is no Ag85A expression of Mycobacterium tuberculosis or BCG. The report and patent application of the simultaneous overexpression of Ag85B protein in BCG

Method used

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  • Recombination BCG vaccine rBCG::AB
  • Recombination BCG vaccine rBCG::AB
  • Recombination BCG vaccine rBCG::AB

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Construction and Identification of Recombinant Plasmid pMAg85AB

[0037] Molecular biology techniques were performed routinely: 1.8kb Ag85A (fbpA, Rv3804c) and 1.5kb Ag85B (fbpB, Rv1886c) genes were respectively amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR. The amplification conditions were all 95°C for 5min, then 94°C for 1min, 60°C for 1min, 72°C for 2min, 30 cycles, and finally 72°C for 10min. PCR products were recovered using the AxyPrep PCR Product Recovery Kit (Axygen). The 1.8 kb fbpA gene was digested with BamHI and XbaI, and the 1.5 kb fbpB gene was digested with BamHI and HindIII, and recovered with AxyPrep DNA Gel Extraction Kit (Axygen), respectively. Then the fbpA gene was connected with the pcDNA3.1(-) recovered by the same enzyme digestion; the fbpB gene was connected with the pcDNA3.1(+) recovered by the same enzyme digestion; fbpA and fbpB were subcloned separately or successively into the same Escherichia coli-mycobacterium Sh...

Embodiment 2

[0040] Establishment of Recombinant BCG rBCG::AB

[0041] The preparation of recombinant BCG rBCG::AB is as follows. First prepare the competent state of BCG. Take 1ml of BCG strain in the logarithmic growth phase, inoculate aseptically in 50ml of 7H9 liquid medium, and culture at 37°C for 2 weeks. After cooling the medium on ice for 2 hours, the bacteria were collected by centrifugation at 4°C. Add 1ml of 10% ice-cold glycerin to resuspend, and disperse the bacteria with a sticky grinder. After that, wash 3 times with 1 / 2, 1 / 10 and 1 / 50 of the volume of the original culture volume of ice-cold glycerol, and finally resuspend with 1ml of ice-cold glycerol, aliquot into 100μl tubes, and store at -80°C for later use. The second is the electroporation of BCG. First, add 100 μl of competent BCG strain to a pre-cooled 2mm Bio-Rad electroporation cup, add the purified recombinant plasmid pMAg85AB (less than 5 μl), mix well, and ice-bath for 10 minutes, remove the air bubbles in t...

Embodiment 3

[0043] Cellular Immunity Characteristics of Recombinant BCG rBCG::AB

[0044] 10 respectively 6 CFU recombinant vaccines rBCG::AB, rBCG::85B, rBCG::85A and rBCG::261 were used to immunize C57BL / 6 mice, and PBS was used as control. Six weeks and 24 weeks after the mice were immunized, the spleen lymphocytes of the immunized mice were aseptically isolated, and the number of secreted IFN-γ cells specific to the spleen lymphocyte antigen was detected by ELISPOT technique. Antigens include PPD, Ag85A and Ag85B proteins, the antigen concentration is 2 μg / ml, and the number of cells is 10 6 . See image 3 , the recombinant vaccine rBCG::AB induced short-term and long-term increases in the number of secreted IFN-γ cells specific to Ag85A and Ag85B proteins in spleen lymphocytes.

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Abstract

The invention provides a recombination BCG vaccine rBCG::AB for protecting against tubercle bacillus and a preparation method thereof. The preparation method comprises the following steps: firstly, amplifying full-length genes containing promoters and signal peptides of Ag85A and Ag85B proteins, and establishing a recombination colibacillus-mycobacteria shuttle expression plasmid after subcloning; and then, transforming the plasmid in a BCG vaccine to form the recombination BCG vaccine rBCG::AB which can simultaneously excessively express the Ag85A and Ag85B proteins. The recombination BCG vaccine rBCG::AB realizes the high-abundance overexpression of the Ag85A and Ag85B proteins in the culture filtrate of the recombination BCG vaccine. After the immunization of animals, the recombination BCG vaccine rBCG::AB can obviously induce cellular immune response to the Ag85A and Ag85B proteins and provide stable and persistent protection against infection, thereby overcoming the defects of short protection time, instable protection against adult tuberculosis and the like of the traditional BCG vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant BCG vaccine against Mycobacterium tuberculosis and a preparation method thereof. Background technique [0002] Tuberculosis (TB) is one of the important public health problems seriously affecting human health worldwide. In recent decades, the incidence of TB has been on the rise, and about 2 million people die from TB every year. The number of people infected with Mycobacterium tuberculosis (Mycobacterium tuberculosis) in China has reached 550 million, and the number of patients and deaths of tuberculosis ranks first among infectious diseases. [0003] Bacillus Calmette-Guerin (M.bovis BCG, BCG) is the only vaccine clinically used to immunize infants and young children to prevent tuberculosis. It is an attenuated live vaccine obtained by repeated in vitro subculture of Mycobacterium bovis (M.bovis) in 1921. , is currently used in 161 countries and regions ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21A61K39/04A61P31/06C12R1/32C12R1/19
Inventor 范雄林王春石春薇付瑞玲陈玲霞周志广卢佳陈振华方正明
Owner HUAZHONG UNIV OF SCI & TECH
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