C2株蓝氏贾第鞭毛虫ILP基因的克隆表达及蛋白结构分析

目的 利用大肠杆菌对C2株蓝氏贾第鞭毛虫ILP蛋白(Impact-like protein)进行克隆表达,运用生物信息学软件进行蛋白结构分析。方法 提取C2株蓝氏贾第鞭毛虫基因组DNA,PCR扩增ILP基因,构建pGM-T重组载体,挑选阳性克隆并进行序列分析;将ILP基因连入原核表达载体pET-28a(+),并转化大肠杆菌Rosetta(DE3),IPTG诱导表达。运用PSIPRED和SWISS-MODEL进行蛋白结构分析。结果 成功构建了原核表达载体pET-28a(+)-ILP,该基因全长831 bp。SDS-PAGE结果显示,目的蛋白条带出现在相对分子量约33 kD的位置,与预期相符。Western blot结果表明,大肠杆菌成功表达了重组蛋白。结论 成功克隆、表达并分析C2株蓝氏贾第鞭毛虫ILP蛋白,为蓝氏贾第鞭毛虫ILP蛋白结构与功能的研究提供了有价值的资料。

Cloning,expression and protein structural analysis of ILP gene from C2 Giardia lamblia

The aim of this study is to clone, express and analyze ILP (Impact-like protein) gene from C2 Giardia lamblia. Genome DNA of C2 Giardia lamblia was extracted with DNA extraction kit. Specific primers were designed by primer design software Primer Premier 5. ILP gene was amplified by PCR and cloned into pGM-T vector, and transformed into E. coli (Top10) competent bacteria cells. Positive clone was chosen and the sequence was analyzed. The pGM-T-ILP recombinant vector was digested by restriction endonuclease NcoⅠand XhoⅠ. Target gene was restructured into pET28a(+) vector at the NcoⅠand XhoⅠrestriction sites to generate recombinant expression vector pET28a(+)-ILP. The vector was transformed into E. coli Rosetta (DE3) and induced with IPTG (isopropyl β-D-thiogalactopyranoside). SDS-PAGE result showed the objective protein band in the position of the relative molecular weight was 33 kDa. Western blot analysis with His-Tag antibody indicated that the ILP gene was successfully expressed in the prokaryotic Rosetta (DE3). Our results provided valuable experimental data for further understanding the structure and function of ILP gene.