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- 2018
犬弓首蛔虫卵黄原蛋白DUF1943结构域的克隆及原核表达
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Abstract:
为克隆犬弓首蛔虫Toxocara canis，T. canis卵黄原蛋白DUF1943结构域(The domain of unknown function 1943，DUF1943)(Tc-duf1943)，构建其原核表达载体pET-28a(+)/duf1943，并检测重组蛋白在大肠杆菌Escherichia coli，E. coli中的表达形式.该文根据T. canis的转录组数据，以T. canis雌、雄虫cDNA为模板扩增Tc-duf1943，然后与表达载体pET-28a(+)连接，转化E. coli BL21(DE3)，经IPTG诱导，对目的蛋白的原核表达形式和蛋白质纯化进行鉴定.结果显示Tc-duf1943序列长度为864 bp，含有一个完整的开放阅读框，编码288个氨基酸；SDS-PAGE电泳结果表明，重组蛋白以包涵体形式存在，相对分子质量大小约为3.5×104；对表达条件进行优化后，以0.4 mmol/L IPTG，于37 ℃诱导4 h时可获得大量目的蛋白；利用Ni-NTA亲和层析柱纯化可以获得具有较高纯度的目的蛋白.该试验成功构建了pET-28a(+)/duf1943原核表达载体，为进一步研究TcDUF1943的生物学功能奠定了基础.
To clone the DUF1943 (the domain of unknown function 1943) of vitellogenin in Toxocara canis, the prokaryotic expression vector pET-28a (+) /duf1943 of Tc-duf1943 was constructed and its expression form in Escherichia coli was detected. According to the transcriptome data about T. canis, and using the cDNA of female and male as templates, Tc-duf1943 was cloned successfully. Then the target gene was connected with the prokaryotic expression vector pET-28a(+) (pET-28a(+)/duf1943), E. coli BL21 (DE3) was transformed and the prokaryotic expression and purification of the protein was identified. The results showed that Tc-duf1943 was 864 bp in length, with a complete open reading frame, encoding 288 amino acids, and SDS-PAGE indicated that the recombinant protein was expressed in inclusion bodies, with a molecular weight of about 3.5×104, The optimal condition of prokaryotic expression was induced by 0.4 mmol/L IPTG for 4 hours at 37 ℃. Purified with Ni-NTA affinity chromatography, target protein with high purity was obtained. In conclusion, this experiment successfully constructed the prokaryotic expression vector of pET-28a(+)/duf1943, which laid a foundation for further biological function study of TcDUF1943
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