东北黑熊源粪肠球菌EfaA基因的克隆表达及生物信息学分析

摘要

为检测熊源粪肠球菌心内膜炎抗原(EfaA)基因,用于快速检测东北黑熊粪肠球菌感染病例,本试验根据GenBank登录的粪肠球菌EfaA基因序列(登录号:U03756.1)合成了1对PCR引物,通过PCR法扩增合成长度为689 bp的目的片段.将PCR产物克隆于pMD19-T载体,之后将其转入大肠杆菌DH5α感受态细胞筛选阳性克隆.提取扩增后的重组质粒pMD19-T-EfaA并进行PCR、酶切鉴定、序列测定及蛋白质结构预测.结果显示,本试验成功克隆出熊源粪肠球菌EfaA基因,与GenBank登录的ATCC 29212菌株同源性为100.0％.本试验成功构建了重组克隆载体pMD19-T-EfaA,并对测序结果进行了蛋白质结构的预测,为进一步建立黑熊粪肠球菌病的诊断方法提供了理论依据,同时也为黑熊疾病的防制奠定了基础.%In order to detect Enterococcus faecalis endocarditis antigen(EfaA)of bear that was used to disclose the infected Northeast Black bear immediately,a pair of PCR primers was synthesized by EfaA gene of Enterococcus faecalis that was recorded in GenBank(accession number: U03756.1)and the target fragment was 689 bp.The PCR product was cloned by pMD19-T vector,then was transferred to Escherichia coli DH5α competent cells and the positive clones were filtered.Recombinant plasmid(pMD19-T-EfaA)was extracted.Identification of PCR and digestion,sequencing,the structure prediction of proteins were operated.The results showed that EfaA gene was cloned successfully,and the gene homology with ATCC 29212 was 100.0％.pMD19-T-EfaA was constructed successfully,structure of proteins was predicted.This study provided theoretical basis for diagnostic methods of Enterococcus faecalis and laid basis for prevention and control of Northeast Black bear disease.