Prokaryotic Expression Of Enterococcal Surface Protein Of Swine-originated Enterococcus Faecalis And Effect Of LPXTG-like Substances On The Biofilm Formation

Enterococci are Gram-positive cocci.They exist mainly in the intestines of humans and animals as normal microflora, and also widely distribute in environments, among them E. faecalis and E.faecium are predominant. Enterococci are mainly used as fungicides, leavens and prebiotics earlier in food or fodder. The drug-resistant bacteria strains was found,because of Enterococcal infections in hospitals increased and overuse of antibiotics, their pathogenicity has been taken more and more seriously.Enterococcus is an important opportunistic pathogen. They can cause severe infection when immunity is low. It is one of the important pathogens that cause hospital-acquired infections. Enterococcus has the ability to form a biofilm (Bacterial biofilm, BF), so Enterococcal infections increased and difficult to treat. BF is not only to provide a good shelter for the bacteria and the body’s defense system and anti-bacterial drugs is difficult to clear. Depth studys of Enterococcus faecalis BF and its mechanism are important to the prevention and treatment of infections. Enterococci biofilm formation is a complex dynamic process and a variety of related genes involve in this process. Enterococcus surface protein (Enterococcal Surface Protein) play an important role in the process of Enterococcus biofilm formation.Through the study of esp, the relationship in the expression of Esp、As、Ace、Gel and biofilm formation in Enteroccus faecalis, which will obtained expression of the success and provide the basis for preparation of monoclonal antibody and development of enterococcus vaccine as well as further studing in pathogenetic mechanisms.Specific primers of Esp were designed according to N fragment of Esp gene sequences included in GenBank. Taking the swine-originated enterococcus faecalis as the template, the 948bp target gene segment was obtained by amplification through PCR, and the ligation of purified product of PCR with cloning vector pTG19-T, was transferred into DH5α-the colony strain. Then the recombinant plasmids were identified by PCR and alyzing of their sequences. Esps of 43 isolates were amplified. 16 isloates of the 43 isolates have esp gene, therefore the carrier rate of Esp gene reached at a low number 37.2%.Esp protein gene was amplified and subcloned into the prokaryotic expression vector pET-32a and constructed pET-32a-esp expression plasmid recombinant. By IPTG induced about 53.5KD fusion protein was obtained.9 isolates of Enterococcus faecalis were divided into biofilm group and non-biofilm group.The luminance ratios of esp、as、ace and gelE of Enterococcus faecalis in biofilm and non-biofilm group were detected by Real Time-PCR.And the expression of esp、as、ace、gelE genes in different groups were detected by real-time PCR and were relatively quantitated though 2-△△Ct method. Moreover,the relevancies between that four genes andbiofilm formation in Enterococcus faecalis were analyzed respectively. The expression of as in biofilm group were 64 times more than the non-biofilm group.The expression level of gelE in biofilm group were 1/8 times less thanthe non-biofilm group.The results showed that the biofilm formation in Enterococcus faecalis was promoted by as,and inhabited by gelE.In the adhesion experiment, the enterococcus faecalis was cultured on 37℃ or 46℃ respective, acted with the different concentrations of positive serum,acted with different methods, and then adhesion to the IPEC-J2. The results indicated that enterococcus faecalis can express Ace on 46℃,and the serum with anti-Ace antibody can prohibit the enterococcus adhesion to the IPEC-J2.But the better method is to make the serum combined with enterococcus faecalis in advance.The enterococcus faecalis was cultured on 46℃, divided into two groups to determine the ability of biofilm formation on abiotic material by. HN9, HN10, HN41 in the two groups was no biofilm formation, while HN12 has a biofilm formation in two groups, but no significant differences. The results show that the Ace does not play a role in Enterococcus faecalis biofilm formation,and biofilm formation is promoted by other proteins and genes.