Construction of sRNA-deficient and-overexpressing strains of Yersinia pestis / 军事医学

Objective To construct small RNA deletion and overexpression strains with a length of less than 100 nt in Yersinia pestis.Methods Deletion mutants of the target sRNAs were constructed by increasing the length of homologous regions.Meanwhile, the high copy plasmid pBAD/HisA was modified into an inducible transcriptional vector as an sRNA-overexpression plasmid by using QuikChange lightning site-directed mutagenesis kit .The presence , size, and transcription-al initiation sites of the indicated sRNA were predicted by transcriptome sequencing , primer extension , and previous stud-ies.The full-length DNA fragments of target sRNAs were transformed into the transcriptional vector .The overexpressing strains of sRNAs were identified by Northern Blot .Results and Conclusion Four sRNAs deletion mutants of sR01, sR02, sR03 and HmsA and three sRNAs overexpression mutants MicF , HmsA and CpxQ were successfully constructed .A method of construction of sRNA deficient and overexpressing strains of Y.pestis has been quickly and efficiently established by λ-Red homologous recombination technology and QuikChange ? lightning site-directed mutagenesis kit.

Construction and preliminary study of biological functions of O157∶H7 typeⅢsecre-tion system effector NleF gene knockout mutant / 军事医学

Objective To construct Escherichia coli O157∶H7 T3SS effector NleF gene knockout mutant and its com-plementary strain, and probe its effects on bacterial growth and cell death .Methods T3SS Effector NleF gene knockout mutant ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 was measured every hour , and the growth curve was drawn .HeLa cells were infected with three kinds of strains , the supernatant of LDH release was detected with cytotoxicity detection kit ,and the cytotoxicity was calculated .Results ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/NleF infected cells.Conclusion EHEC O157∶H7 T3SS Effector NleF has no significant effect on bacterial growth ,but might inhibit host cell death caused by bacterial infection .