熊源粪肠球菌EfaA基因的原核表达

doi:10.16431/j.cnki.1671-7236.2018.01.009

摘要/Abstract

摘要：

试验旨在构建EfaA基因的原核表达质粒，并对其进行表达。根据GenBank中粪肠球菌EfaA基因序列（登录号：U03756）设计合成1对引物，利用PCR法扩增、克隆EfaA基因，并进行生物信息学分析，将EfaA基因亚克隆于pGEX-4T-1原核表达载体，构建pGEX-4T-EfaA原核表达质粒，转化大肠杆菌BL21（DE3）感受态细胞，用IPTG诱导表达，筛选最佳诱导时间并分析其表达蛋白的生物学特征。结果发现，试验成功获得大小为873 bp的粪肠球菌EfaA基因。经IPTG诱导后，获得分子质量约为59 ku的EfaA重组蛋白，以37℃、0.5 mmol/L IPTG诱导6 h表达量最大。经Western blotting分析发现，具有较好的反应原性。本试验成功构建原核表达质粒pGEX-4T-EfaA，并在大肠杆菌BL21（DE3）感受态细胞中成功表达。

关键词: 黑熊; 粪肠球菌; 心内膜炎抗原; 原核表达

Abstract:

This study was aimed to construct and express the prokaryotic expression plasmid of EfaA gene. One pair of PCR primers was designed according to the EfaA gene sequence from GenBank (accession No.:U03756). EfaA was used as target gene to clone and analyze by biological information. EfaA gene was sub-cloned into pGEX-4T-1, PGEX-4T-EfaA prokaryotic expression plasmid was constructed, and the plasmid was transformed into E.coli BL21(DE3), which was induced by IPTG. The best induction time was screened and biological characteristics of the protein was analyzed. The results showed that 873 bp fragment of Enterococcus faecalis EfaA gene was obtained successfully. Induced expression was operated by IPTG and 59 ku EfaA recombinant protein was obtained. The best way of this react was 6 h in 37℃ by IPTG (0.5 mmol/L). The Western blotting result showed good reactogenicity. The prokaryotic expression plasmid pGEX-4T-EfaA was successfully constructed and expressed in E.coli BL21 (DE3).

Key words: Black bear; Enterococcus faecalis; endocarditis antigen; prokaryotic expression

中图分类号:

S852.61⁺1

楚熹橦, 秦晓东, 鲁承, 孙福亮. 熊源粪肠球菌EfaA基因的原核表达[J]. 《中国畜牧兽医》, 2018, 45(1): 71-76.

CHU Xitong, QIN Xiaodong, LU Cheng, SUN Fuliang. Prokaryotic Expression of EfaA Gene of Enterococcus faecium from Bear[J]. , 2018, 45(1): 71-76.

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