Preliminary Study On The Construction And Function Of Mycobacterium Tuberculosis Rv2346c Gene Knock Out Strain

Objective:?1?To construct of Mycobacterium tuberculosis Rv2346c knockout strain with mycobacteriophage recombination system.?2?To investigate the effect and relative molecular mechanism of recombinant protein Rv2346c on murine RAW264.7macrophage and human U937 cells.?3?To observe and investigatethe the virulence and function of Rv2346c gene in infected mice lung tissues treated with Mycobacterium tuberculosis?MTB?standard strain H37Rv,attenuated strain H37Ra,knockoutstrain?Rv2346c,andreplenishingstrain?Rv2346c/?Rv2346c::p MV261.Methods:?1?The affinal exchange sites?AES?of the target gene was built,and then integrated into the phage genomes of M.tuberculosis for harvesting the phagemids.The phagemids were introduced into Mycobacterium smegmatis to get recombinant phages with the same AES.A high titer of the recombinant phages were harvested through amplification in vitro.The M.tuberculosis was transfected and coated on solid medium with hygromycin resistance and cultured for 4 weeks at 37^oC.Single clone was picked out and gene knock-out was confirmed by PCR.?2?DNA synthesis,gene amplification,vector construction,induced expression and protein purification were used to synthesize recombinant protein Rv2346c.Cell Counting Kit-8?CCK8?kit was applied to tested the proliferation of RAW264.7 and U937.Colony formation unit was observed to estimate the growth of BCG.Enzyme-linked immuno sorbent assay?ELISA?was utilized to detect the level of tumor necrosis factor-??TNF-??and interleukin?IL?-6 in co-culture supernatant.Western blot was conducted to measure the expression of NF-?B?nuclear transcription factor-kappa B?p65.T test was applied to compare the means of two independent groups and P<0.05 was considered statistically significant.?3?To verify the function of Rv2346c in MTB infection,C57BL/6 mice were infected intratracheally with MTB strains H37Rv,H37Ra,?Rv2346c,?Rv2346c/?Rv2346c::p MV261.The mortality of each group was observed at the 56^thday after infection.Mice were killed 35 days after infection,lung tissue was obtained aseptically,and a series of dilutions of tissue homogenate were plated on Middlebrook 7H11 agar and bacterial CFU were counted after 4-6 weeks of incubation at 37°C.The lung tissues were collected and the pathological damage of lung tissue induced by MTB was evaluated by detecting the expression of TNF-?and IL-6.Results:?1?The products of PCR and inserted fragment sizes were consisted with the expectation and confirmed to be the target gene.The target fragment of Rv2346c was removed successfully.?2?Recombinant protein Rv2346c was verified by DNA sequencing and Western blot;Rv2346c alone did not affect RAW264.7 or U937 cell proliferation;but promoted the BCG-induced inhibition of proliferation in RAW264.7 or U937 cell?P<0.05?and reduced RAW264.7-medicated immunological killing effect against BGG?P<0.05?;Rv2346c also suppressed the secretion of TNF-?and IL-6 in RAW264.7 or U937 cells?P<0.05?and expression of NF-?B p65?P<0.05?.?3?The survival curve showed higher survival rate in C57BL/6mice treated with?Rv2346c strain for 56 days than that in standard H37Rv strain and the replenished strain?Rv2346c/?Rv2346c::p MV261 strain.After 35 days of MTB infection,the bacterial load of mice treated with knockout strain was much lower than that observed in the standard or replenished strain?P<0.05?.Pathological results of lung tissue showed that the lung tissues in PBS,BCG and H37Ra groups were basically normal,and many inflammatory lesions were observed in the lung parenchyma in standard and replenishing strains,but it was less severe in knockout strain than that in the two other strains.Morphometric analysis of histological sections of HE staining showed that the inflammatory areas of lung tissues in mice treated with the standard strain and the replenishing strain were basically the same,but the inflammatory areas were significantly lighter in knockout strain compared with the standard strain and the replenishing strain,Immunofluorescence results showed that the F4/80 population distributions in the standard strain group and the knockout strain group were generally similar,but it showed higer TNF-?and IL-6fluorescence intensity in knockout strain group compared with that in the other two groups.Conclusions:Rv2346c gene knockout strains of MTB mediated by bacteriophageis are successfully constructed;Recombinant protein Rv2346c can inhibit macrophage-medicated immunological killing effect in RAW264.7 and U937cells infected with BCG.In addition,Rv2346c knockout can significantly reduce the toxicity of MTB in vivo,and the mechanism may be associated with cytokine secretion of TNF-?and IL-6 and activation NF-?B p65 in macrophages inhibited by Rv2346c,thereby inhibiting its cellular immune function,which in turn affects the host's clearance of MTB.