东亚飞蝗细胞色素P450基因的克隆及表达分析
Cloning, expression of cytochrome P450 from Locusta migratoria manilensis
陈义昆1,邬玉兰2,李荔1,连国云2,刘志刚1,2**
点击：2508次 下载：27次
DOI：
作者单位：1.深圳大学生命科学学院深圳518060；2.深圳大学医学院深圳518060
中文关键词：东亚飞蝗， 细胞色素P450， 基因克隆， 表达
英文关键词：Locusta migratoria manilensis, cytochrome P450, gene clone, expression
中文摘要：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pET28a中，在大肠埃希菌Escherichia coli Rosetta中用异丙基βD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（SDSPAGE）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pET28aP450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。
英文摘要：To clone the gene of cytochrome P450 (CYP450) from Locusta migratoria manilensis (Meyen)，produce its recombinant
protein.The cDNA of CYP450 was cloned using specific primers from the total RNA of L. m. manilensis. The cloned gene was
inserted into pMD18T vector and digested by BamH I and Hind III. The cDNA was sequenced and subcloned into pET28a
expression vector. The cloned CYP450 cDNA gene was expressed in Escherichia coli Rosetta by IPTG induction. Result showed
that the cloned cDNA ORF sequence contained 1 551 bp and encoded 516 amino acids. Its sequence homology with the published
one (Accession no. HM153426) was 99% at nucleotide level. The CYP450 was highly expressed in E. coli Rosetta as a unsoluble
protein mainly with the molecular weight of about Mr 53 000 under induction of IPTG.