Application of mycobacterium tuberculosis Rv3833 gene in preparation of preparation for treating mycobacterium tuberculosis latent infection
A technology of mycobacterium tuberculosis and mycobacteria, applied in gene therapy, microbe-based methods, antibacterial drugs, etc., can solve the problems of inaccurate quantification and low resolution, and reduce false positive rate and false positive Effects of interference and improvement of detection sensitivity
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Embodiment 1
[0038] Example 1: Under anaerobic and normal conditions, high expression of the Mycobacterium tuberculosis Rv3833 gene can inhibit the growth of Mycobacterium tuberculosis
[0039] 1. Construction of Mycobacterium tuberculosis Rv3833 Gene High Expression Plasmid
[0040] Using the Mycobacterium tuberculosis H37Rv genome as a template, the Rv3833 gene was amplified by PCR and cloned into the Escherichia coli-Mycobacterium tuberculosis shuttle plasmid pMV261 (such as figure 1 shown), transformed into Escherichia coli DH5a competent cells, the transformation solution was spread on a solid LB medium plate containing 0.05 mg / ml kanamycin, cultured upside down at 37°C, and positive clones were screened. Pick a monoclonal colony, inoculate into 3ml liquid LB medium containing kanamycin, and shake overnight at 37°C. The plasmid was extracted from the bacterial liquid for sequencing, and the plasmid with correct sequencing was the constructed plasmid pMV261-Rv3833 with high expression...
Embodiment 2
[0043] Example 2: Recombinant BCG constructed with Mycobacterium tuberculosis Rv3833 gene
[0044] 1. Construction of recombinant BCG
[0045] Using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template, the Rv3833 gene was amplified by PCR and cloned into the Escherichia coli-Mycobacterium tuberculosis shuttle plasmid pMV361 (such as image 3 shown). After BCG recovery, inoculate into 400ml 7H9 (add 10% ADC and glycerol) medium and culture for 10-14 days. In the logarithmic growth phase, add 100ml of 20% glycine to a final concentration of 4%, continue to cultivate for 24 hours, and break the glass beads Bacterial membranes were washed with 10% glycerol to prepare BCG-competent cells. Transfer the competent cells into the electroporation cup, and perform electroporation at 2.5kv / cm, 25μF, 1000 ohms, add 15ml of 7H9 (adding 10% ADC, glycerol and Tween80) medium to the transformation solution, cultivate overnight at 37°C, and centrifuge Take 125 μl of the ...
Embodiment 3
[0052] Embodiment 3: the recombinant subunit vaccine constructed with the Mycobacterium tuberculosis Rv3833 gene
[0053] 1. Construction of recombinant plasmids
[0054] Using the Mycobacterium tuberculosis H37Rv strain genomic DNA as a template, the Rv3833 gene was amplified by PCR and cloned into the eukaryotic expression plasmid vector pcDNA3.1+ (such as Figure 4 shown), sequenced and identified. Preparation of E.coli DH5α competent cells. The correctly identified pcDNA-Rv3833 gene recombinant plasmid was transformed into competent cells, cultured in LB medium at 37°C overnight, and the large amount of recombinant plasmid was extracted using Qiagan's endotoxin-free plasmid mass extraction kit;
[0055] 2. Analysis of immune protection of recombinant subunit vaccine
[0056] The above-mentioned subunit vaccine was immunized to BalB / c mice once every two weeks for a total of 3 times. At the same time, the PBS group was set as a control. Four weeks after the last immuni...
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