Cloning And Bait Vectors Construction Of Ami、auto、inlB、inlC、 InlJ Of Listeria Monocytogenes Virulence Genes

Listeria monocytogenes（LM） is an important foodborne pathogenic bacteria. Itcan cause gematosepsis, pregnant miscarriage and cerebromeningitis in human andanimals. In the cource of LM infection, many virulence factors which functionsdepend on the combination with the effector cells. So far, the specific mechanism ofcerebromeningitis which is caused by LM is not clear. So, the virulence genes Ami,Auto, InlB, InlC, InlJ of LM were cloned, sequenced, constructed bait plasmids, andthen detected expression and self-active of bait plasmids in this study, the constructionof bait expression vectors is the basic step for yeast two-hybrid. This provides ascientific basis for revealing the mechanism of bait proteins in the process of LMinfecting brain and then clarifying pathogenic mechanism of LM. The main researchcontents and results are as follows:1.Cloning and sequence analysis of Ami, Auto, InlB, InlC, InlJ of LMvirulence genes: In order to investigate the variation of virulence genes (Ami, Auto,InlB, InlC and InlJ) of Listeria monocytogenes serotype4b LM90isolated fromclinical diseased sheep in Xinjiang. The open reading frams (ORFs) of five genes’were amplified by PCR and cloned into pMD19-T vector, positive colonies werescreened and sequenced. These gene sequences of Ami, Auto, InlB, InlC and InlJwere submitted to GenBank (accession number: KF826613, KF826614, KF826611,KF703749, KF826612) and analyzed by DNAMAN. Alignment results indicated thatthe homology of the five virulence genes was98.04%,96.53%,98.20%,96.41%, or98.23%at nucleotide sequence level, and99.73%,99.83%,98.67%,99.66%or99.76%at amino acid sequence level between LM90and F2365, respectively. Ourresearch results revealed that the five tested virulence genes of Listeriamonocytogenes LM90are relatively stable.2.Construction and function assay of bait plasmids: In order to screen hostprotein which were interacted with five genes(Ami, Auto, InlB, InlC, InlJ) by yeasttwo-hybrid system, the five bait plasmids were constructed. Firstly, five clonevectors(Ami, Auto, InlB, InlC, InlJ) and bait vectors(PDHB1, PBT3-N) were ligatedafter purification and digested with sfiⅠ, and then transformed into DH5ɑ. The fiverecombinant bait plasmids were identified by PCR and digesting wih sfiⅠ, then fivebait plasmids(Ami-PDHB1,Auto-PDHB1, InlB-PDHB1, InlC-PDHB1, InlJ-PDHB1)were transformed into yeast (NMY51) by LioAC method. Secondly, to detectexpression of the bait plasmids in the yeast.(1) The western blot was used to detectexpression of bait protein, which were incubated with anti-LM90rabbit antibody andsheep anti-rabbit IgG-HRP.(2) The positive vector (pOst1-NubI) and negative vector(pPR3-N) was transformed into yeast carrying bait plasmids, respectively, thendetected the expression of bait plasmids by colony-counting method on SD-Trp-Leu,SD-Trp-Leu-His and SD-Trp-Leu-His-Ade plates. The results showed that the baitplasmids and positive yeasts were obtained according to PCR amplification and digestion with sfiⅠ. The bait protein were not detected by Western blot. But in thefunction assay, there were white or pink colonies on selective plates in the positivecontrol group, There were fewer colonies or nothing on SD-Trp-Leu-His andSD-Trp-Leu-His-Ade plates in the negative group, the results indicated that the baitplasmids were successfully expressed.3.Screening3-AT concentration to inhibit self-activation of bait-bearing yeast:In order to inhibit self-activation of bait plasmid and reduce the false positive in yeasttwo-hybrid, it’s necessary to screen the optimal3-AT (a competitive inhibitor of theHis gene) concentration. The cDNA library vector pPR3-N was transformed into thebait-bearing yeast by LioAc method. The bait-bearing yeasts growth were obserevedon SD-Leu-Trp-His-Ade plates which3-AT concentrations was10M,15M,20M,25M,30M,35M,40M,45M, respectively. The reasults showed that the bestconcentrations of3-AT of Ami-PDHB1, Auto-PDHB1, InlB-PDHB1, InlC-PDHB1,InlJ-PDHB1was45M,25M,30M,20M,20M, respectively.