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本文克隆了东亚飞蝗Locusta migratoria manilensis(Meyen)细胞色素P450(cytochrome P450)基因全长,表达重组蛋白,并对其可溶性进行了分析。通过提取东亚飞蝗总的RNA,反转录成cDNA,设计特异性引物,PCR克隆东亚飞蝗细胞色素P450基因,将测序正确的目的片段克隆至原核表达载体pET-28a中,在大肠埃希菌Escherichia coli Rosetta中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测重组蛋白表达结果。结果表明:东亚飞蝗细胞色素P450基因开放阅读框全长为1 551 bp,编码516个氨基酸,与GenBank中已登录的东亚飞蝗细胞色素P450基因(HM153426)的同源性为99%,重组质粒pET-28a-P450在E.coli Rosetta中获得高效表达,重组蛋白相对分子质量(Mr)约为53 000,主要以包涵体的形式存在。
In this study, we cloned the full-length cytochrome P450 gene from Locusta migratoria manilensis (Meyen) and expressed the recombinant protein, and analyzed its solubility. By extracting total RNA of Locusta migratoriae and reverse transcribing into cDNA, specific primers were designed and PCR was used to clone Cytochrome P450 gene from Locusta migratoria manilensis. The correct fragment was cloned into prokaryotic expression vector pET-28a, Expression in Escherichia coli Rosetta was induced with isopropyl-β-D-thiogalactoside (IPTG). Recombinant protein expression was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the open reading frame of cytochrome P450 gene of Locusta migratoria manilensis is 1 551 bp in length and encodes a protein of 516 amino acids with a homology of 99% with that of the previously reported Cytochrome P450 gene (HM153426) in GenBank. The plasmid pET-28a-P450 was highly expressed in E.coli Rosetta. The relative molecular mass (Mr) of the recombinant protein was about 53 000, which was mainly in the form of inclusion body.