摘要
目的建立抗药性相关糜蛋白酶基因稳定转染细胞系。方法采用PCR方法,以淡色库蚊抗药性品系cD NA文库为模板,扩增出抗药性品系高表达的糜蛋白酶基因编码区;经T/A克隆测序鉴定后,亚克隆到真核表达载体pIE13,构建重组昆虫细胞表达载体pIE13/chy,经酶切和PCR鉴定后,与带有筛选标记的pIE1neo质粒共转染蚊细胞C6/36,通过G418选择培养,建立转染细胞系;用RT PCR、Westernblotting鉴定糜蛋白酶基因的转录表达。结果成功构建了真核表达载体pIE13/chy,证明糜蛋白酶基因已转入C6/36细胞,建立了稳定转染细胞系,表达产物分子质量单位约30ku,与理论值相符。结论稳定转染细胞系的建立和基因表达为进一步研究糜蛋白酶基因与抗药性的关系奠定了基础。
Objective To construct a stable-transfected cell line of the chymotrypsin gene from deltamethrin resistant Culex pipiens pallens. Methods The coding region of chymotrypsin gene expressed highly in deltamethrin resistant strain of Cx. 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 chymotrypsin gene was subcloned into the eukaryotic expression vector pIE1-3, and so the recombinant expression vector of insect cell (pIE1-3/chy) was constructed. Identifications of restriction digestion and PCR were made before the above recombinant vector was co-transfected into Aedes albopictus C6/36 cell with pIE1-neo plasmid. A stable-transfected strain was established through screening culture by G418 and the transcription and expression of the chymotrypsin gene was identified by RT-PCR and Western blotting. Results It was confirmed that the eukaryotic expression vector was successfully constructed, and that chymotrypsin gene was transfected into C6/36 cell and a stable-transfected cell line was established. The expressed chymotrypsin protein about 30 ku was dectected by Western blotting. Conclusion The construction of the stable-transfected cell line and the expression of the gene will provide solid experimental foundation for further studies on the relationship between the chymotrypsin gene and the insecticide resistance.
出处
《中国寄生虫病防治杂志》
CSCD
2005年第2期81-84,共4页
Chinese Journal of Parasitic Disease Control
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(No.03KJB180082)
江苏省资源生物技术重点实验室开放研究课题资助项目。
关键词
糜蛋白酶基因
昆虫细胞表达载体
抗药性
基因转染
C6/36细胞
Chymotrypsin gene
expression vector for insect cell
insecticide resistance
gene transfection
C6/36 cell
