Study On The Biological Characteristics And Genetic Traits Of The New Strain Of Tuberculosis Vaccine

Objective: To investigate the fusion strains(B/R strain) growth characteristics, colonization and hereditary constructed by Bacillus Calmette Guerin(BCG) and Mycobacterium tuberculosis H37 Ra strains(H37Ra).Methods: 1. BCG, H37 Ra and B/R strains were cultured in Sauton Broth Medium respectively, and measured the OD values of bacteria BCG, H37 Ra and B/R strains in different cultured time of the 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days at a wavelength of 600 nm. And then with the time(Time) as the horizontal coordinates, OD600 value for the vertical coordinates, draw the above three strains growth curve. 2. C57BL/6 mice were randomly divided into 3 groups, and vaccinated subcutaneously with B/R strains, BCG and H37 Ra bacteria liquid 0.1ml(including the number of living bacterium was 1.0 ×106). After immunized 15, 30, 45, 60 and 75 days, the spleen, lung and liver of mice were taken into and made into viscera tissue homogenate, take evenly serous 0.1ml inoculated on the surface of the Roche solid medium slant and cultured for 20 days, count of three groups of strains in different organs of the colony forming units(Log10cfu). 3. The total RNA of B/R strains, BCG and H37 Ra were extracted, purified and reverse transcription into c DNA, after using SYBR GreenⅠQuantitative Real-time PCR(q RT- PCR) respectively to detect the m RNA expression levels of esat-6, ag85 b, mpt70 and mpt83 gene in the above three strains. To compare and analyze the differences of esat-6, ag85 b, mpt70 and mpt83 gene expression level in three different strains. 4. B/R strains were extracted from the first generation to the twentieth generation of genomic DNA, the esat-6, ag85 b, mpt70 and mpt83 genes were amplified for each generation of B/R strains by using the template PCR, and compared and analyzed the stability of the B/R strains.Results: 1. According to the draw a growth curve, compared with BCG and H37 Ra strains, B/R strains growth period for adaptation is long, and 6 days after began into the logarithmic phase, and 27 days into the stable platform. 2. Under the same immune time, the Log10 cfu of B/R strains in different organs of mice were higher than those of BCG and H37 Ra strains. Among them, compared with the BCG and H37 Ra strains, the Log10 cfu of B/R strains in the liver of mice were slightly higher, but the difference was not statistically significant(P >0.05). And with the increase of immune time, three strains of Log10 cfu in the liver of mice were up and down, the time trend is not obvious; In five different immune time, the Log10 cfu of B/R strain in the spleen of mice were significantly higher than those in BCG strains group, the difference is statistically significant(P <0.05); Immune after 15, 60 and 75 days, the Log10 cfu of B/R strain in the spleen of mice than the H37 Ra strains group, the difference is statistically significant(P <0.05). With the increasing time of immunization, the Log10 cfu of threestrains in the spleen of mice showed a downward trend overall. Compared with 60 days, the Log10 cfu of H37 Ra and B/R strains in the spleen of mice has a smaller increase in the 75 days after immunization, but the Log10 cfu of BCG in the spleen of mice showed a downward trend. In five different immune time, the Log10 cfu of B/R strain in the lungs of mice were significantly higher than those in BCG strains group, the difference is statistically significant(P <0.05); Immune after 15 days and 45 days, the Log10 cfu of B/R strains in the lungs of mice than H37 Ra strains group, the differences have statistical significance(P <0.05). And with the increase of immune time, the overall Log10 cfu of the three strains in mice lungs showed a downward trend. 3. According to the result of q RT-PCR, compared with BCG strains, the m RNA relative expression levels of mpt70 and mpt83 genes in B/R strains were down regulated, the difference was not statistically significant(P >0.05); The m RNA relative expression levels of mpt70 and mpt83 genes in H37 Ra strains were also down regulated, but the difference was statistically significant(P <0.05); Compared with H37 Ra strains, the m RNA relative expression levels of mpt70 and mpt83 genes in B/R strains and BCG strains were increased, the difference was statistically significant(P <0.05); The m RNA relative expression levels of esat-6 and ag85 b genes in B/R strains also were increased, and the difference was statistically significant(P <0.05) 4. The results of PCR showed that esat-6, ag85 b, mpt70 and mpt83 genes of B/R strain first to twentieth generation were amplified, and the size in the same position, clear and bright bands.Conclusions: 1. The growth cycle of B/R strain was 30 days, and could survive in mice for at least 75 days, and the colonization ability was stronger. 2. B/R strains in the 20 generations of the gene level can achieve the quality requirements of the stability of biological products.