大劣按蚊卵黄蛋白原基因cDNA全长序列扩增及功能域蛋白的表达与纯化研究  

Amplification of full-length cDNA sequence of Anopheles dirus vitellogenin gene and expression and purification of its functional domain

作　　者：于莎莎 胡辉 秦杰 王静[1] 刘婷[1] 刘婷婷 王英[1] YU Shasha;HU Hui;QIN Jie;WANG Jing;LIU Ting;LIU Tingting;WANG Ying(Department of Tropical Medicine,College of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Critical Care Medicine,the PLA 74th Group Army Hospital,Guangzhou,Guangdong Province,510220,China)

机构地区：[1]陆军军医大学(第三军医大学)军事预防医学系热带医学教研室,重庆400038 [2]中国人民解放军陆军第七十四集团军医院重症医学科,广州510220

出　　处：《第三军医大学学报》2021年第12期1140-1145,共6页Journal of Third Military Medical University

基　　金：重庆市自然科学基金项目(cstc2018jcyjAX0182);国家自然科学基金(81702035,81971971)。

摘　　要：目的对大劣按蚊卵黄蛋白原(Anopheles dirus vitellogenin, AdVg)基因cDNA的全长序列进行扩增和分析,并对其功能域蛋白进行表达与纯化。方法基于前期克隆的AdVg基因cDNA片段,利用RACE PCR技术,扩增AdVg基因的5′末端及3′末端序列。用Vector NTI软件将测序得到的AdVg cDNA片段、5′末端序列、3′末端序列进行校正,拼接得到完整的AdVg cDNA全长序列。对AdVg全长序列进行功能域预测。利用pet28a(+)构建重组质粒进行AdVg功能域蛋白的表达与纯化。结果成功获得AdVg基因全长cDNA序列,包括117 bp的5′UTR序列、192 bp的3′UTR序列和6 186 bp的ORF序列。AdVg蛋白主要包含Vit N、DUF1943及vWD 3个结构域。通过构建重组质粒,成功表达和纯化了AdVg Vit N功能域蛋白。结论成功克隆了大劣按蚊卵黄蛋白原基因的全长序列,并获得了重组蛋白。Objective To amplify and analyze the full-length cDNA of Anopheles dirus vitellogenin(AdVg) gene, and express and purify its functional domain. Methods Based on the cDNA fragments of AdVg gene cloned earlier, the full-length sequence was obtained through rapid amplification of cDNA ends(RACE). The sequences of AdVg cDNA fragment, 5′ end and 3′ end were corrected and assembled to obtain a complete AdVg cDNA full-length sequence by Vector NTI software. Then the full-length sequence of AdVg was used to predict the functional domain. Finally, recombinant plasmid pet28 A(+) was used to express and purify the AdVg functional domain. Results The full-length cDNA sequence of the AdVg gene was successfully obtained, including a 117 bp 5′ UTR sequence, a 192 bp 3′UTR sequence, and a 6 186 bp open reading frame(ORF) sequence. Meanwhile, the analysis showed that the AdVg protein mainly contained 3 structural domains, including Vit N, DUF1943 and vWD. Then we successfully expressed and purified the functional domain AdVg Vit N by constructing a recombinant plasmid. Conclusion The full-length cDNA of AdVg is successfully cloned, and the recombinant protein of its functional domain Vit N is obtained in this study.

关 键 词：大劣按蚊 卵黄蛋白原基因 RACE扩增 蛋白表达与纯化 

分 类 号：Q962[生物学—昆虫学] Q963