淡色库蚊抗药性相关胰蛋白酶基因稳定转染细胞系的建立与表达鉴定  

Construction and expression identification of a stable-transfected C6/36 cell line of deltamethrin resistance associated trypsin gene from Culex pipiens pallens

作　　者：公茂庆[1] 顾燕[1] 胡小邦[1] 孙艳[1] 马磊[1] 李秀兰[1] 朱昌亮[1] 

机构地区：[1]南京医科大学病原生物学系,江苏省现代病原生物学重点实验室,南京210029

出　　处：《中国人兽共患病杂志》2005年第6期454-457,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(No.30170835);江苏省高校自然科学基金资助项目(03KJB180082)

摘　　要：目的为进一步研究胰蛋白酶基因与抗药性的关系。方法采用PCR方法,以淡色库蚊抗性品系cDNA文库为模板,扩增出抗药性品系高表达的胰蛋白酶(trypsin)基因编码区。T/A克隆测序鉴定后,亚克隆到昆虫细胞表达载体pIE13,构建重组昆虫细胞表达载体pIE13/try。经酶切和PCR鉴定后,与带有筛选标记的pIE1neo质粒共转染蚊虫细胞C6/36,通过G418选择培养,建立稳定转染细胞。用RTPCR、Westernblotting鉴定胰蛋白酶基因的转录表达。结果酶切和PCR鉴定表明,成功构建昆虫细胞表达载体pIE13/try;证实胰蛋白酶基因已转入C6/36细胞,并建立稳定转染细胞系,成功地表达目的基因。结论稳定转染细胞系的建立和基因表达为进一步研究胰蛋白酶与抗药性的关系提供了良好的实验基础。In order to research the relationship between the trypsin and the insecticide resistance,the coding region of trypsin, expressed highly in resistance strain of Culex pipiens pallens was amplified by PCR from the cDNA library constructed in resistance strain of the mosquito. After identification of T/A clone and sequencing, the trypsin gene was subcloned into the eukaryotic expression vector pIE1-3, and the recombinant expression vector of insect cell (pIE1-3/try) was constructed. Identifications of restriction digestion and PCR were carried out before the above recombinant vector was co-transfected into Aedes albopictus C6/36 cell with pIE1-neo plasmid.After screening culture by G418, a stable-transfected strain was established, and the transcription and expression of the trypsin gene were identified by RT-PCR and Western blotting. It was confirmed that the eukaryotic expression vector was successfully constructed by restriction digestion and PCR, and that trypsin gene was transfected stably into the C6/36 cell and the trypsin protein was expressed successfully. And it was demonstrated that a stable-transfected cell line was constructed. In conclusion the construction of the stable-transfected cell line and the expression of the gene of interest provide solid experiment foundation for further studies on the relationship between the trypsin and the insecticide resistance.

关 键 词：胰蛋白酶基因 昆虫细胞表达载体 抗药性 基因转染 C6/36细胞 

分 类 号：R384.1[医药卫生—医学寄生虫学]