Recombinant BCG vaccine rBCG::X

A technology of recombinant BCG vaccine and BCG vaccine is applied in the fields of genetic engineering vaccines and tuberculosis vaccines, which can solve problems such as difficulty in obtaining HspX protein, and achieve the effects of strong immune protection type and strong protection effect.

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

Problems solved by technology

However, it is difficult to obtain a large amount of HspX protein. In the previous work, we established an Escherichia coli genetic engineering system and purification technology for overexpressing HspX protein, and applied for a national invention patent (National Invention Patent Application No. 200710051915.5), laying the foundation for the application of this patented vaccine pre-basis

Method used

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  • Recombinant BCG vaccine rBCG::X
  • Recombinant BCG vaccine rBCG::X
  • Recombinant BCG vaccine rBCG::X

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Construction and Identification of Recombinant Plasmid pMHspX

[0039] The molecular biology technique was carried out according to the routine: the 0.8kb HspX (acr, Rv2031c) gene was amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR technique. The amplification conditions were as follows: 95°C for 5 minutes, then 94°C for 45s, 60°C for 45s, 72°C for 50s, 30 cycles, and finally 72°C for 5 minutes. PCR products were recovered using the AxyPrep PCR Product Recovery Kit (Axygen). After digesting the 0.8kb acr gene with BamHI and XbaI, it was recovered with AxyPrep DNA Gel Extraction Kit (Axygen). Then the acr gene was ligated with the pcDNA3.1(-) recovered by the same enzyme digestion, and then subcloned into the Escherichia coli-mycobacterium shuttle plasmid pMV261 to form the recombinant plasmid pMHspX. Further enzyme digestion identification and sequence analysis proved that the constructed recombinant expression plasmid was completely correct. Seq...

Embodiment 2

[0041] Establishment of Recombinant BCG rBCG::X

[0042] The preparation of recombinant BCG rBCG::X 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 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 pMHspX (less than 5 μl), mix well, and ice-bath for 10 minutes, remove the air bubbles in the cup...

Embodiment 3

[0044] Cellular Immunity Characteristics of Recombinant BCG rBCG::X

[0045] 10 respectively 6 CFU recombinant vaccines rBCG::X 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, Ag85B and HspX proteins, the antigen concentration is 2 μg / ml, and the number of cells is 10 6 . See image 3 , the recombinant vaccine rBCG::X induced an increase in the number of secreted IFN-γ cells specific to the HspX protein in the spleen lymphocytes of short-term and long-term immunized mice.

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Abstract

The invention provides an anti-tubercle bacillus recombinant BCG vaccine rBCG::X. The recombinant escherichia coli-mycobacteria shuttle plasmid containing a coded HspX protein gene is transformed into the BCG vaccine to form the recombinant BCG vaccine rBCG::X; the recombinant BCG vaccine rBCG::X realizes the over expression of the HspX protein, can obviously induce the organism to generate the cellular immune response aiming at the HspX protein after animal immunity, and generate stable and endurable anti-infective protection, thus overcoming the shortcoming that the BCG vaccine has short protection period and unstable protection effect on the adult tuberculosis and the like.

Description

technical field [0001] The invention belongs to the fields of genetic engineering vaccines and tuberculosis vaccines. Specifically, the present invention provides a novel recombinant BCG against Mycobacterium tuberculosis, that is, the gene (acr, Rv2031c) of the HspX protein of Mycobacterium tuberculosis or BCG is cloned into BCG, and overexpressed in BCG to form recombinant BCG:: X. 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] BCG is currently the only preventive vaccine against tuberculosis, but its protection period is short and its protective ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21A61K39/04A61P31/06C12R1/32C12R1/19
Inventor 范雄林石春薇卢佳陈振华陈玲霞周志广王春付瑞玲方正明
Owner HUAZHONG UNIV OF SCI & TECH
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