关键词: 结核分枝杆菌, 基因敲除, Lrp/AsnC家族蛋白, Rv2779c基因

Abstract:

Objective To explore the function of Mycobacterium tuberculosis （Mtb） Rv2779c gene， an Rv2779c deletion mutant was constructed. Methods Using the genomic DNA of the Mtb reference strain H37Rv as the template， a 500 bp upstream and downstream fragment of Rv2779c gene for homologous recombination was amplified by PCR， and then constructed into the shuttle plasmid p0004s. The homologous recombinant plasmid p0004s was constructed into temperature-sensitive phasmid phAE159 and then packaged as phage. After the phage infection， the homologous recombinant element was integrated into the H37Rv genome. The positive clones were screened by hygromycin resistance marker in the recombinant element. The resistance-positive clones were identified by PCR. The growth differences between Rv2779c deletion mutant and H37Rv WT strain in 7H9 complete medium and high concentration rifampicin medium were compared. Results The recombinant plasmid p0004s-ΔRv2779c containing the upstream and downstream sequence of Rv2779c homologous recombinant arm was successfully constructed， and the plasmid was ligated with phAE159 which contained mycobacterium temperature sensitive element to obtain the recombinant phasmid. Finally， the successfully packaged phage infected H37Rv and the resistance positive clone was selected. PCR results showed that the Rv2779c gene of Mtbwas knocked out successfully. The knockout of Rv2779c had little effect on the growth of Mtb in normal medium， but significantly reduced the survival of Mtb in high concentrations of rifampin. Conclusion The Rv2779c deleted strain of Mtb was successfully constructed， which lays an important foundation for the functional study of Rv2779c gene. And preliminary studies revealed that this gene affected the growth of Mtb in rifampicin-containing medium.

Key words: mycobacterium tuberculosis, gene knockout, Lrp/AsnC family, Rv2779c gene