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根据家蝇CYP6A1、CYP6D1以及致倦库蚊CYP6E1的保守区氨基酸序列设计一组简并引物,采用RT-PCR的方法,从白纹伊蚊四龄期活体幼虫总RNA中扩增出与设计相符的基因片段。利用T-A克隆法,将获得的基因片段克隆人pGEM-T easy载体。经限制性内切酶酶切和PCR鉴定证明重组成功。将筛选的阳性克隆经序列测定及同源性分析,表明共获得CYP6家族中9个的新cDNA序列。为进一步研究细胞色素P450的多样性及其与抗药性形成的分子机制打下基础。
A set of degenerate primers was designed based on the conserved amino acid sequences of housefly CYP6A1, CYP6D1 and CYP6E1 of Culex quinquefasciatus. RT-PCR was used to amplify the total RNA from the live larvae of fourth instar of Aedes albopictus in accordance with the design Of the gene fragment. The obtained gene fragment was cloned into human pGEM-T easy vector by T-A cloning method. Restriction endonuclease digestion and PCR identification proved successful recombination. The positive clones screened by sequence analysis and homology analysis showed that a total of 9 CYP6 family of new cDNA sequences were obtained. It laid the foundation for further study on the diversity of cytochrome P450 and its molecular mechanism of drug resistance formation.