白念珠菌烯醇化酶N端肽段ENO1-319P的原核表达和鉴定

Expression and identification of N-terminal fragment of Candida albicans enolase

目的制备白念珠菌烯醇化酶N端基因重组肽段ENO1-319P并检测其抗原性。方法选择与人烯醇化酶蛋白α-ENO同源性低的白念珠菌烯醇化酶肽段ENO1-319P,将其编码DNA序列克隆到表达载体pET28a(+),并在大肠埃希菌BL21(DE3)中用IPTG诱导带有His标签的重组肽段的表达。用SDS-PAGE电泳分析,TALON金属亲和层析柱纯化重组蛋白,其抗原性用侵袭性白念珠菌感染(ICAI)患者血清进行western blot评估。结果成功表达并纯化了目的肽段,该蛋白可与ICAI患者血清发生特异性反应。结论获得了白念珠菌烯醇化酶N端肽段ENO1-319P的基因表达工程菌株,为将此重组肽段用于制备特异性更高的抗体打下基础。

Objective To prepare the recombinant N-terminal fragment ENO1-319P of Candida albicans enolase and evaluate its antigenicity.Methods The DNA fragment encoding ENO1-319P,which showed low homology to the α-ENO1 of Homo sapiens enolase,was cloned into the prokaryotic expression vector pET28a（＋）.The recombinant peptide with His6 tag was then produced by the induction of IPTG in E.coli.BL21（DE3）,purified by TALON metal affinity resins,and analyzed with SDS-PAGE.The immunogenicity of the recombinant peptide was evaluated by western blot with sera of patients with invasive candidiasis.Results The recombinant peptide was successfully expressed and purified.Western blot showed that the recombinant protein could specifically react with the sera from systemic candidiasis patients.Conclusion The engineering bacteria strain expressing recombinant N-terminal fragment of Candida albicans enolase was prepared successfully for the research on the recombinant peptide with high specificity.