淡色库蚊抗性转录因子FTZ-F1蛋白原核表达与鉴定

Prokaryotic expression and identification of the resistant transcription factor FTZ-F1 in Culex pipiens pallens

目的 对淡色库蚊抗性转录因子FTZ-F1蛋白进行原核表达并鉴定。方法 对转录因子FTZ-F1蛋白进行生物信息学分析,包括蛋白的理化性质,二级结构及三维同源模型。采用PCR技术扩增FTZ-F1基因,比对分析后选取FTZ-F1基因保守区和功能区片段,片段长度为1122bp,然后将其连接至pET-32a(+)载体,构建重组质粒pET-32a(+)-FTZ-F1,再转化入感受态细胞BL21(DE3)后用IPTG诱导重组质粒pET-32a(+)-FTZ-F1的表达,运用SDS-PAGE和Western blot初步鉴定重组蛋白FTZ-F1的表达,采用His标签镍离子蛋白纯化柱对表达产物进行纯化和增加蛋白的量,用SDS-PAGE和Western blot鉴定重组蛋白FTZ-F1的表达。结果 FTZ-F1蛋白有744个氨基酸,相对分子质量为79.57×10^(3)。1%琼脂凝胶鉴定PCR扩增为单一条带,大约为2 240 bp,测序结果表明重组质粒pET-32a(+)-FTZ-F1构建正确。采用SDS-PAGE检测重组蛋白FTZ-F1,结果表明蛋白FTZ-F1在原核表达系统中主要以可溶性形式表达,其相对分子质量约为38.9×10^(3)。Western blot检测重组蛋白FTZ-F1,采用抗His标签抗体识别FTZ-F1,结果表明为单一条带。纯化后的蛋白浓度为1.25 mg/ml,纯度为90%。结论 成功构建了重组质粒pET-32a(+)-FTZ-F1,鉴定重组蛋白FTZ-F1的表达,为该蛋白生物学功能的研究奠定了基础。

Objective To create a system for prokaryotic expression and identify the expressed product of FTZ-F1,a transcription factor related to deltamethrin resistance in Culex pipiens pallens. Methods the FTZ-F1 gene was analyzed by bioinformatics, including physical and chemical properties, secondary structure and three-dimensional homology model. The conserved and functional fragment of FTZ-F1 gene with a length of 1 122 bp was amplified and ligated to pET-32a(+)vector to construct the recombinant plasmid pET-32a(+)-FTZ-F1. Then the recombinant plasmid was transformed into competent cell BL21(DE3) and its expression was induced with 0.5 mM IPTG. The expressed product was purified through Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blotting. Results Bioinformatics analysis showed that FTZ-F1 had 744 amino acids and the molecular weight was 79.57×10^(3). 1% agarose gel electrophoresis and sequencing indicated that the fragment of FTZ-F1 gene was successfully amplified and the recombinant plasmid pET-32a(+)-FTZ-F1 was constructed correctly. The expression of the recombinant protein FTZ-F1 was mainly in a soluble form in the prokaryotic expression system by SDS-PAGE,and its molecular weight was about 38.9×10^(3). The purified protein was characterized as a single band by Western blotting using anti-His tag antibody and its concentration was about 1.25 mg/ml, the purity was about 90%. Conclusion The recombinant plasmid pET-32a(+)-FTZ-F1 was successfully constructed and the expression of the recombinant protein FTZ-F1 was identified and purified, which laid a foundation for the study of the biological function of the protein.