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根据报道的十几种昆虫CYP4家族基因的氨基酸序列保守区域设计一对引物,利用RT-PCR技术扩增编码青杨脊虎天牛Xylotechus rusticus中肠细胞色素氧化酶CYP4G2蛋白的cDNA片段,构建原核表达载体pET-CYP4G2,将其转化入大肠杆菌Escherichia coli JM109中表达。序列分析结果表明,该基因(CYP4G2,GenBank登录号为EF429250)保守区域阅读框全长387bp,编码129个氨基酸残基,预测分子量和等电点分别为16.9kD和5.75;推导的氨基酸序列与已报道的昆虫CYP4家族氨基酸序列一致性较高(63%~86%),且具有细胞色素氧化酶的典型特征。IPTG诱导后,SDS-PAGE电泳检测到一条22kD大小的外源蛋白,与预测融合蛋白的分子量大小相应。CO差光谱分析证明重组菌表达了有活性的pET-CYP4G2。
A pair of primers was designed according to the reported conserved region of CYP4 family of dozens of insects. RT-PCR was used to amplify the cDNA fragment encoding the CYP4G2 protein of midgut of Xylotechus rusticus The expression vector pET-CYP4G2 was transformed into E. coli Escherichia coli JM109 for expression. Sequence analysis showed that the conserved region of this gene (CYP4G2, GenBank accession number EF429250) was 387 bp in length and encoded 129 amino acid residues. The predicted molecular weight and isoelectric point were 16.9 kD and 5.75, respectively. The deduced amino acid sequence was The CYP4 family of amino acid sequences of the reported insects are highly consistent (63% -86%) and have the typical characteristics of cytochrome oxidase. After induced by IPTG, a 22 kD foreign protein was detected by SDS-PAGE electrophoresis, corresponding to the predicted molecular weight of the fusion protein. CO differential spectroscopy demonstrated that the recombinant bacteria expressed the active pET-CYP4G2.