摘要：
目的 利用分枝杆菌噬菌体重组系统构建结核分枝杆菌R v2346 c基因敲除株,体内感染小鼠肺组织,观察R v2346 c基因的毒力,从而探讨结核分枝杆菌R v2346 c基因功能.方法 构建同源交换位点,随后将其整合到结核分枝杆菌噬菌体基因组,获得噬菌粒,将噬菌粒导入耻垢分枝杆菌,获得整合有同源交换位点的重组噬菌体,经体外扩增获得高滴度的重组噬菌体,对结核分枝杆菌进行转染,将转染后的结核分枝杆菌涂布到潮霉素抗性的固体培养基,37°C培养4周,挑取单克隆,通过PCR验证基因敲除成功,将野生株(wild type,WT)及敲除株(knockout,KO)结核分枝杆菌感染C57BL/6J小鼠肺组织,6～8周后观察小鼠死亡率、小鼠肺组织炎性反应程度及肺组织结核分枝杆菌体外培养菌落形成数.两组间比较采用配对t检验,率的比较采用χ2检验.结果 经鉴定PCR产物及插入片段大小与预期值相符,且为所需目的基因片段,成功切除靶片段,体内感染小鼠6～8周,KO株感染小鼠肺组织后,体外菌落形成数明显低于WT株感染(15.0±0.8比90.0±1.5,t=23.0361,P<0.05),小鼠肺组织炎性反应程度明显轻于WT株感染(1040±89比1960±56,t=7.1016,P<0.05),其小鼠总死亡率明显低于W T株感染(53％ 比20％,χ2=6.1112,P<0.05).结论 成功构建了噬菌体介导的结核分枝杆菌R v2346 c基因敲除株,为R v2346 c基因功能的研究奠定了基础.%Objective To investigate the Rv2346c gene function through constructing Rv2346c gene knockout strains of Mycobacterium tuberculosis (M . tuberculosis) mediated by bacteriophage and observing its virulence after infecting mice lung tissue in vivo .Methods 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 was imparted into Mycobacterium smegmatis to get recombinant phages with the same AES .A high titer of the recombinant phages was 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°C .Single clone was picked out and gene knock-out was confirmed by PCR . Then C57BL/6J mice were infected with either wild type strain (WT ) or knockout strain (KO ) of M . tuberculosis .Mice mortality ,lung tissue inflammation and colony-forming units (CFU ) counts in vitro were observed 6 to 8weeks post infection with different strains . Paired-samples t test was used for comparison between groups ,chi-square test was used for comparison of rates .Results 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 and the mice were infected for 6-8 weeks .Themice infected with Rv2346c KO strain had reduced mortality (53％ vs 20％ ,χ2 =6 .1112 ,P<0 .05) ,lung tissue inflammation (1040 ± 89 vs 1960 ± 56 ,t=7 .1016 ,P<0 .05) and CFU count in vitro (15 .0 ± 0 .8 vs 90 .0 ± 1 .5 ,t=23 .0361 , P<0 .05) compared with WT strain 6-8 weeks post infection .Conclusion Rv2346c gene knockout strains of M . tuberculosis mediated by bacteriophageis are successfully constructed ,which establishes the foundation for the future gene function study of Rv2346c .
