| Enterococci are Gram-positive cocci which are aerobic or facultativeanaerobic organisms, globular or orbicular-ovate, catalase negative, no spore andoften occur in single, pairs or chains. They exist mainly in the intestines of humansand animals as normal microflora, and also widely distribute in environments such assoil, water and so on, among them E. faecalis and E.faecium are predominant.Enterococci are mainly used as fungicides, leavens and prebiotics earlier in food orfodder. And their pathogenicity has been taken more and more seriously, as theirantibiotic resistence is recognised.Enterococci infect and make people and animals ill mainly through adhesion,invasion and tissue damages, among which the adhesion is the first and the mostimportant step.Since it is only by adhesion on the cell or cell-extracellular matrix thatcan lead to final tissue lesion. Among the virulence factors of enterococci, adhersin ofcollagen from enterococcus faecalis (Ace) is obviously serious in the early infectionof enterococci. Through the study of ace, detailed reference datas are expected for thefuture rapid diagnosis of enterococci and development of effective vaccines, whichwill guarantee the quality and safety of animal food.Specific primers of Ace were designed according to the A fragment of Ace genesequences included in GenBank. Taking the swine-originated enterococcus faecalis asthe template, the813bp target gene segment was obtained by amplification throughPCR, and the ligation of purified product of PCR with cloning vector pTG19-T, wastransferred into DH5α--the colony strain.Then the recombinant plasmids wereidentified by PCR, restriction endonucleases digestion and analyzing of theirsequences. Aces of three standard strains which have been preserved in our laboratoryand43isolates were amplified. And all the three isolates have Ace gene, and so with30isloates of the43isolates, therefore the carrier rate of Ace gene reached at a highernumber--71.7%. At the same time, homology of the46isolates were compared andthe rate reached at the level of98.0%--100%,which was corresponded with the valueof97.5%or greater that we have studied. The results indicted that ace widely existed in Enterococci and highly conserved, so detection and prevention for Enterococcicould be conducted using this protein.Then production of the PCR connected with the expression vector pET-32a, andthe recombinant plasmid was transferred into the expression bacterial BL21, after thePCR of the plasmid, restriction enzyme digestion and sequencing, it’s identified theconstruction of the recombinant plasmid was successful. Induced for6h by the IPTGwith the concentration of0.8mmol/L on37℃,it prokaryotic expressed dissolubleprotein. We got the antibody with high level after using the protein immunsing therabbits. It’s identified that the protein had best immunogenicity after western blot. Inthe adhesion experiment, the enterococcus faecalis was cultured on37℃or46℃respective, acted with the positive or negative serum, and then adhesion the IPEC-J2.The results indicated that enterococcus faecalis both can express Ace on37℃or46℃respective, but the quantity on46℃is larger than it on37℃,and the serum withanti-Ace antibody can prohibit the enterococcus adhesion the IPEC-J2.After the optimization of all conditions, we established an accurate and effectiveindirect ELISA method using the Ace and the positive serum. The optimal conditions:the concentration of the Ace which coaching the wells was1μ/mL, the optimaldilution of the serum was1:800,the coaching time was under37℃for2h and then4℃overnight, the blocking agent should be1%BSA,the optimal blocking time was2h, the best reaction time of the serum was1h,the HRP-antibody’s optimal dilutionwas1:600, the best reaction time of the second antibody was also1h,the optimalaction time with the substrate was15min, the critical value of OD was0.082, whenP/N≥2.1,and the OD≥0.082,we could decide the serum was positive. According thespecificity and reproducibility experiments, the indirect ELISA method which Iestablished could effectively detect the positive and negative serum. |
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