编码日本血吸虫Argonaute蛋白全长cDNA克隆、表达及初步鉴定  

Cloning,expressing and identifying of a full-length cDNA encoding Argonaute from Schistosoma japonicum

作　　者：杨燕萍[1,2] 郭素霞[1] 陈晶[1] 林矫矫[1] 刘宗平[2] 程国锋[1] 

机构地区：[1]中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室,上海200241 [2]扬州大学兽医学院,扬州225009

出　　处：《中国人兽共患病学报》2010年第9期830-834,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30901068);上海市科学技术委员会项目(10410703400);上海市人才发展基金(2009032);中央级公益性科研院所基本科研业务费项目(2008JB18和2009JB08)

摘　　要：目的获得编码日本血吸虫Argonaute基因的全长cDNA,并进行克隆和重组融合蛋白表达。方法利用RACE技术获得编码日本血吸虫Argonaute基因的全长cDNA并对其进行生物信息学分析。利用实时荧光定量PCR(Realtime PCR)分析该基因在日本血吸虫中的转录情况。原核表达保守功能域蛋白和全长蛋白,利用保守功能域重组融合蛋白免疫昆明系小鼠制备多克隆抗体。免疫印迹(Western blot)进行免疫学分析。结果本研究获得了一个编码日本血吸虫Argo-naute基因的全长cDNA,其完整开放阅读框为3 030 bp,编码1 009个氨基酸,预测分子量为111.7 kDa。生物信息学分析表明该基因编码的蛋白序列具有Argonaute家族蛋白的典型结构域特征,且与人、鼠Argonaute蛋白具有较高的同源性。Real-time PCR分析表明该基因在7,14日龄的日本血吸虫童虫中转录较高。Western blot分析制备的多抗具有较高的特异性并能识别全长融合蛋白。结论获得了编码日本血吸虫Argonaute基因的cDNA及重组蛋白,为进一步研究该蛋白的功能奠定了基础。The present study was intended to obtain the full-length cDNA encoding Argonaute protein（SjAGO） in Schistosoma japonicum and subsequently express the recombinant protein in E.coli BL21.A cDNA fragment encoding Argonaute was harvested as applying the RNA template isolated from 14 day schistosomula by using 5＇RACE technique.Upon bioinformatical extension,a full-length cDNA encoding Argonaute protein from Schistosoma japonicum was obtained including a complete Open Reading Frame.The Argonaute protein has 1009 amino acids with 111.7kDa molecular weight predicted.Bioinformatical analysis indicated that the SjAGO contains PAZ and PIWI domain,which is a typical character for Argonaute protein family in other organism such as mice and human.The transcript profile of SjAGO from egg,7,14,22,32 and 42d schistosomes was determined by real-time PCR and showed that the transcript of SjAGO was predominantly observed at schistosomula stage.The cDNA fragment encoding PIWI domain was further cloned into a pET28b（＋） vector and the recombinant plasmid was transfected into E.coli BL21cell.The recombinant protein was purified by NTA column.Kunming mice were immunized with the purified protein to raise polyclonal antibodies.The full-length cDNA encoding SjAGO was also cloned into a pET32a（＋） vector to express the recombinant protein.The full-length SjAGO expressed in E.coli BL21 was successfully validated when applying the polyclonal antibodies by using Western blot.In conclusion,a full-length cDNA encoding Argonaute protein from Schistosoma japonicum was cloned and some preliminary studies were also carried out.Results presented here provide foundational information for further investigation on Argonaute＇s roles in schistosome development.

关 键 词：日本血吸虫 Argonaute蛋白 RACE 功能分析 

分 类 号：R383.2[医药卫生—医学寄生虫学]