Abstract:To prokaryotic express prokaryotically and to purify the efaA protein from Enterococus faecalis so as to provide the basis for the further study on the pathogenesis and clinical sero-diagnosis of endocarditis caused by E.faecalis,efaA gene of E.faecalis was amplified by PCR,the PCR-amplified product was digested with restriction enzymes and cloned into prokaryotic vector pET32a to construct the recombinant plasmid pET30a/efaA.This recombinant plasmid was confirmed by double enzyme digestion with BamhI and Xhol and then subjected to sequencing,and transformed to E.coli BL21(DE3).Expression of the fusion protein was induced by IPTG,and analyzed by SDS-PAGE and Western blotting.The recombinant fusion protein was purified by His-binding affinity chromatography.It was shown that efaA gene of 943 bp in size was amplified from Enterococus faecalis and the recombinant plasmid pET30a,/efaA was successfully constructed and expressed in E.coli BL21.The purified product was found to be 34 kDa in molecular weight as demonstrated by SDS-PAGE and Western blotting.It is evident that the efaA protein of E.faecalis can be successfully expressed and purified.
[1]Creti R,I mperi M,Bertuccini L,et al.Survey for virulence de-terminants amongEnterococcus faecalisisolated from different sources[J].Med Microbiol,2004,53:13-20.
[2]Olesen HV,Moiler JR.Enterococcal infections:clinical findings and treat ment considering antibiotic resistance in Denmark[J].Ugeskr Laeger,2002,164(18):2386-2390.
[3]Mart nez OP,Mu oz SJ,Arriola MP,et al.Enterococcal infec-tive endocarditis:description of12cases[J].An Med Interna,2007,24(11):539-542.
[4]Zhang L,Nguyen J,Epel man S,et al.Enterococcal endocarditis presenting as an isolated aortic valve aneurysm:case report and reviewof literature[J].J Am Soc Echocardiogr,2008,21(12):1391-1396.
[5]Larry M,Baddour.Virulence factors among gram-positive bacte-ria in experimental endocarditis[J].Infection and I mmunity,1994,62(6):2143-2148.
[6]Shorrock PJ,Lambert PA,Aitchison EJ,et al.Serological re-sponse inEnterococcus faecalisendocarditis determined by en-zyme-linked immunosorbent assay[J].J Clin Microbiol,1990,28:195-200
[7]Lowe AM,Lambert PA,Smith AW.Cloning of anEnterococcus faecalisendocarditis antigen:homology with adhesins fromsome oral streptococci[J].Infection and I mmunity,1995,63(2):703-706.
[8]Low YL,Jakubovics NS,Flat man JC,et al.Manganese-depend-ent regulation of the endocarditis associated virulence factor EfaA ofEnterococcus faecalis[J].J Med Microbiol,2003,52(2):113-119.
[9]Lin DP,Wada S,Jimenez-Lucho VE.Enterococcus faecalis endocarditis presenting as meningitis[J].Infection,1998,26(5):304-305.
[10]Bitar FF,Jawdi RA,Dbaibo GS,et al.Paediatric infective endo-carditis:19-year experience at a tertiary care hospital in a devel-oping country[J].Acta Paediatr,2000,89(4):427-430.