摘要
为了对十二指肠贾第虫(简称贾第虫)γ贾第素(γ-giardin)进行原核表达及亚细胞定位,研究其生物学功能,提取贾第虫总RNA,反转录成cDNA,经PCR获得γ-giardin基因片段,克隆至原核表达载体pET-28a(+),构建重组表达质粒pET-28a-γ-giardin,再转化入大肠杆菌Rosseta(DE3)后,用IPTG诱导表达,表达蛋白经SDS-PAGE验证,并用切胶纯化法纯化。针对重组蛋白制备了多克隆抗体,并用ELISA测定其效价,Western-blot分析其免疫原性,并利用免疫荧光定位技术对γ-giardin进行定位。结果表明,成功构建了原核表达质粒pET-28a-γ-giardin。表达的重组蛋白的分子质量约为38 ku,且以包涵体形式存在,纯化后的重组蛋白的纯度高达90%以上。ELISA和Western-blot结果显示,γ-giardin抗体具有良好的抗原特异性与优异的抗原结合活性。免疫荧光定位结果表明γ-giardin主要位于贾第虫滋养体的腹吸盘。本研究为今后探讨贾第虫致病机制、临床诊断和治疗奠定了理论基础。
Prokaryotic expression and subcellular localization of the γ-giardin gene of Giardia duodenalis were conducted to study its biological function.Total RNA of G.duodenalis was extracted from the trophozoites and then reverse-transcribed into c DNA.The γ-giardin gene was amplified using PCR and cloned into prokaryotic expression vector p ET-28a(+) to construct a recombinant expression plasmid p ET-28 a-γ-giardin,and then transformed into Escherichia coli Rosseta(DE3).The expression was induced using IPTG,verified by SDS-PAGE,and purified by the method of gel cutting.Polyclonal antibodies were prepared against recombinant proteins.Protein immunogenicity was determined by ELISA and Western-blot analysis.The localization of γ-giardin was studied by immunofluorescence.The results showed that p ET-28 a-γ-giardin was successfully constructed,had a molecular weight around 38 ku,and expressed mainly in the form of inclusion bodies.The purity of the purified recombinant protein was as high as 90% or more.ELISA and Western-blot analysis showed that γ-giardin antibody had good antigen specificity and excellent antigen binding activity.Immunofluorescence localization technique indicated that γ-giardin was mainly located in the ventral disk of trophozoites.This study provided a theoretical basis for the future investigation of the pathogenesis,clinical diagnosis,and treatment of G.duodenalis.
作者
石东东
张斯雯
朱伟宁
范琳琳
杨萱桐
李巍
SHI Dong-dong;ZHANG Si-wen;ZHU Wei-ning;FAN Lin-lin;YANG Xuan-tong;LI Wei(Heilongjiang Key Laboratory for Zoonosis/College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2019年第8期1019-1024,共6页
Chinese Veterinary Science
基金
国家科技支撑计划项目(2017YFD0501302)
