摘要
为表达多房棘球绦虫钙网蛋白EmCRT,并对其进行初步鉴定及结构与补体结合功能分析。本研究以多房棘球绦虫囊cDNA为模板,通过PCR扩增目的基因,克隆至pcDNA3.3-Myc载体,构建重组质粒pcDNA3.3-Myc-EmCRT;PCR、酶切及测序鉴定其正确后,以脂质体转染法转染Hela细胞,运用Western blot、细胞免疫荧光方法检测重组蛋白在细胞中的表达情况;应用生物学软件分析EmCRT结构与功能特点。结果显示:成功构建了重组质粒pcDNA3.3-Myc-EmCRT,其在Hela细胞中可高效表达重组钙网蛋白,表达产物约为46 kDa;预测该蛋白N端含有1个18 aa的信号肽序列,与其他寄生虫CRT氨基酸序列同源性高达41%~56%,存在4个C1q潜在结合位点。本研究结果为抗包虫疫苗和新药的研制提供重要的理论基础。
To obtain the recombinant Echinococcus multilocularus calreticulin (EmCRT) protein and analyze its structure and complement binding function,cDNA from E.multilocularus;Cyst was used as the template to amplify EmCRT gene by PCR and the amplification products were then subcloned into the pcDNA3.3-Myc plasmid to construct recombinant plasmids.After PCR amplification,restriction enzymes digestion and sequence,the recombinant plasmid DNA was transfected into Hela cell with Lipofectamine^(TM).The expression of recombinant EmCRT in cells was detected by Western blot and immunofluorescence.The structure and function of EmCRT were analyzed by biological software.The results showed that the recombinant plasmid was successfully constructed and EmCRT was highly expressed in Hela cells with a molecular mass of appx.46 kDa.The first 18 amino acids of EmCRT N-terminal formed a signal peptide as predicted by software.In addition,EmCRT shared 41%-56% identity of amino acid sequence of CRTs to other parasites and there were 4 potential binding sites of C1q in EmCRT.In this study,the overexpression recombinant plasmid of EmCRT gene was successfully constructed and 4 potential binding sites of C1q were found,providing an important theoretical basis for the development of anti-hydatid vaccines and new drugs.
作者
陈路娟
程喆
王彦海
赵利美
CHEN Lujuan;CHENG Zhe;WANG Yanhai;ZHAO Limei(Department of Pathogen Biology,School of Preclinical anid Forensic Medicine,Baotou Medical College,Baotou 014060,China;Parasitology Research Laboratory,School of Life Sciences,Xiamen University,Xiamen 361102,China)
出处
《中国动物传染病学报》
CAS
北大核心
2021年第2期40-46,共7页
Chinese Journal of Animal Infectious Diseases
基金
国家自然科学基金项目(81860362)
内蒙古自治区自然科学基金项目(2018BS08013)
内蒙古自治区高等学校科学研究项目(NJZZ18185)
包头医学院博士科研启动基金项目(BSJJ201805)。
关键词
多房棘球绦虫
钙网蛋白
真核表达
补体C1Q
Echinococcus multilocularus
calreticulin
eukaryotic expression
complement C1q
