多房棘球绦虫钙网蛋白的原核表达及初步鉴定  

The prokaryotic expression and preliminary identification of calreticulin of Echinococcus multilocularis(EmCRT)

作　　者：陈路娟 咸思奇 闫艳[1] 徐志坚 程喆[2] 于桂霞[1] 王彦海[2] 赵利美 CH2EN Lu-juan;XI1AN Si-qi;YAN Yan;XU Zhi-jian;CHENG Zhe;YU Gui-xia;WANG Yan-hai;ZHAO Li-mei(Department of Pathogen Biology,School of Preclinical and Forensic Medicine,Baotou Medical College,Baotou,Inner Mongolia 014060,China;Parasitology Research Laboratory,School of Life Sciences,Xiamen University)

机构地区：[1]内蒙古科技大学包头医学院基础医学与法医学院病原生物学教研室,内蒙古包头014060 [2]厦门大学生命科学学院寄生动物研究室

出　　处：《中国病原生物学杂志》2022年第6期654-659,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81860362);包头医学院博士科研启动基金项目(BSJJ201805);包头医学院扬帆计划项目(No.BYJJ-YF201720)。

摘　　要：目的原核表达多房棘球绦虫钙网蛋白EmCRT,并对其免疫学功能进行初步鉴定。方法以多房棘球绦虫囊cDNA为模板,PCR扩增目的基因,克隆至pGEX-4T-2载体,构建重组质粒pGEX-4T-2-EmCRT;PCR、酶切鉴定及测序正确后,转化至表达菌BL21(DE3),并用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达;运用10%SDS-PAGE电泳以及Western blot分析鉴定表达的重组蛋白rEmCRT。结果成功构建了重组质粒pGEX-4T-2-EmCRT,转化表达菌BL21后,经IPTG诱导表达及纯化,获得了纯度较高的可溶性重组蛋白rEmCRT,相对分子质量约为72 ku(含表达载体序列);通过Stains all染色,表明rEmCRT具有Ca^(2+)结合能力;免疫学特性鉴定表明rEmCRT具有较好的抗原特异性。结论成功表达多房棘球绦虫钙网蛋白EmCRT,具有免疫反应性,可为包虫病诊断及其疫苗研发提供理论基础。Objective We construct the recombinant Echinococcus multilocularus calreticulin(rEmCRT)protein by prokaryotic expression and identify its immunological function primarily.Methods cDNA from E.multilocularus vesicles was used as the template to amplify Emcrt gene by PCR and the amplification products were then subcloned into the pGEX-4 T-2 plasmid to construct recombinant plasmids pGEX-4 T-2-EmCRT.After PCR amplification,restriction enzymes digestion and sequence,the recombinant plasmid was transformed into BL21(DE3)and expressed by isopropyl-β-d-thiogalactoside(IPTG)induction.The expression of rEmCRT was detected by 10%SDS-PAGE and Western blot.Results The recombinant plasmid pGEX-4 T-2-EmCRT was transformed into BL21 after it was successfully constructed,and then the recombinant protein rEmCRT was induced to express by IPTG and purified.We obtained the soluble recombinant protein rEmCRT which has a higher purity and the molecular weight was about 72 ku(including the expression vector sequence).The rEmCRT was stained blue by Stains all indicating that its calcium-binding feature.Immunological characterization showed that rEmCRT was a highly immunogenic antigen.Conclusion The recombinant Echinococcus multilocularus calreticulin(rEmCRT)was expressed successfully and it has immunological function which provide an important theoretical basis for the diagnosis of echinococcosis and the development of anti-echinococcosis vaccines.

关 键 词：多房棘球绦虫 钙网蛋白 表达和鉴定 抗原性 

分 类 号：R383.3[医药卫生—医学寄生虫学]