Clonging And Prokaryotic Expression As Well As Bioinformatics Analysis Of Gene From E.Faecalis Strain TLME3

1.Enterococcus faecalis is a new zoonotic pathogen, important pathogens for hospital-acquired infections, death, and Lamb encephalitis gallinarum sepsis and other animal diseases, especially drug-resistant strains caused great difficulties to the prevention and treatment. Enterococcus faecalis infection in the host process is by its Ace-mediated adhesion on the host cell to complete the colonization and infection with the first step. Ace is an MSCRAMM, given to the cell wall anchor protein similar. Ace adhesion of the target substance is the ECM, it is usually the epithelial cells cover the host tissue damage when exposed to bacterial colonization on and lead to infection. Ace crystal structure βI folds to form a groove, and collagen-binding site. Serum and collagen type IV can of Ace mRNA production significantly increase transcriptional regulator Ers-encoded protein is a repressor protein of Ace, QS-Fsr system or GelE expression to interrupt, can significantly enhance the adhesion of Enterococcus faecalis collagen.2. In order to detect whether Enterococcus faecalis strain TLME3contains AceA gene, layings a foundation for expression of AceA protein in prokaryotic cell. One pair of specific primers was designed and synthesized according to the AceA gene sequence from GenBank of the database of NCBI for No. CP002621gene of Enterococcus faecalis in917167～918123bp, the AceA gene was PCR amplificated. After purification and recovery, the products of purification and recovery was connectted with pMD18-T, transformed into the DH5a. Positive clones detected by PCR, recombinant plasmid identificated by single or double restriction enzyme, the correct plasmid send to biological company for sequenceing. Agarose gel electrophoresis of PCR amplification products and recombinant plasmid showed the bands were both about957bp. Single restriction enzyme of pMD18-T-AceA recombinant plasmid by gel electrophoresis was about at3649bp. There were2bands by double enzyme gel electrophoresis, the band of the vector was at2692bp, the band of target gene was at957bp. The results show that, Enterococcus faecalis strains TLME3AceA protein gene was successfully cloned in this experiment.3,In order to further redearch the function of AceA protein from Enterococcus faecalis strain TLME3, measure the immunocompetence, explore the possibility of which as candidate proteins for the vaccine against Enterococcus faecalis septicemia from goslings and biological agents for diagnosis, connected the AceA gene from Enterococcus faecalis TLME3strain with pET-28a (+) prokaryotic expression vector, constructed the recombinant plasmid pET-28a (+)-AceA. After transfered into E. coli BL21(DE3), identified by PCR, enzyme digestion by BamHI, Xhol, and sequencing, expressed under IPTG induction, then detected by SDS-PAGE for the fusion protein. The results showed that there are about957bp strip of PCR product by Agarose Gel Electrophoresis, one strip was6326bp of recombinant plasmid pET-28a (+)-AceA by single enzyme digestion, another two strips containing5369bp of pET-28a (+) and957bp of AceA gene by double enzyme digestion; and there was about40.0KD of target protein by SDS-PAGE. AceA protein from Enterococcus faecalis strain TLME3was expressed efficiently in BL21(DE3) in this experiment.4. Further experimental studies have been made on AceA protein from Enterococcus faecalis strain TLME3, laied a foundation for constructing epitope vaccine for septicemia infection by Enterococcus faecalis from goslings, The AceA gene from TLME3and its proteins sequence were searched in homology with biological-software such as Lasergene7.0, Gene Runner, SWISS-MODEL and BLAST, analyzed by multiple alignment, phylogenetic tree was reconstructed, and variability was predicted;_The physical and chemical characteristics of component, molecular weight, titration curve and isoelectric point from the AceA protein sequence were analyzed; glycosylation sites modification sites and motif of the primary structure were predicted, The potential antigenic epitopes was predicted by comprehensive analysis according to the position of β-turn and random coin of protein secondary structure, plasticity regions, hydrophilic regions, antigenic index (AI) and surface accessibility regions, model of the tertiary structure was predicted, and the homology of AceA with it from other strains were analyzed; mRNA of secondary structure was predicted. The results showed that there were59and88sequence closely related according to the nucleotide and protein sequences respectively, the homology of which as97%～100%and96%～100%, the mutational rate of nucleotides and amino acids were1.31%and1.63%, fall into the2nodes of the same cluster with88proteins, and at the same site with AF260891, in a small branches with ZP05569670and ZP07552605, also closed with the remaining sequences; Molecular weight is40.0kD, containing19kinds of amino acids, the most is Thr, secondly is Glu and Asn, isoelectric point is4.32, which is acidic protein; containing N-matrix glycosylation modification sites; the position of β-turn and random coin was basically the same with ATCC29200; the homology was98.63%with the tertiary structure of AceA protein imitated with SWISS-MODEL; There are8potential antigenic epitopes such as270～284that predicted comprehensively; secondary structure of mRNA contains many kinds of structural elements. The result showed that AceA genes from TLME3was conserved, which can be used as epitope vaccine or protein of diagnostic reagent.