摘要
目的制备、评价特异性抗刚地弓形虫(Toxoplasma gondii)自噬相关蛋白8(autophagy protein 8,TgAtg8)多克隆抗体。方法生物信息学方法分析弓形虫基因组中自噬相关蛋白的基因序列和氨基酸序列。以刚地弓形虫RH株速殖子cDNA为模板,PCR扩增TgAtg8编码基因,克隆入pGEX-6p-1载体,构建pGEX-6p-1-TgAtg8重组质粒,转化至大肠埃希菌(Escherichia coli)BL21。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析重组蛋白表达情况。谷胱甘肽-S-转移酶(GST)标签亲和层析法纯化表达产物。以纯化的TgAtg8蛋白为抗原免疫日本大耳白兔,获得特异性抗TgAtg8多克隆抗体。以制备的多克隆抗体作为一抗,进行Western blotting分析和间接免疫荧光(IFA)检测。结果双酶切和测序结果证实,原核表达质粒pGEX-6p-1-TgAtg8构建成功。SDS-PAGE和Western blotting结果表明,TgAtg8蛋白在E.coli BL21中获得高效表达,其融合蛋白相对分子质量(Mr)约40 000,与理论值相近。Western blotting结果证实,所制备的多克隆抗体能特异性识别融合蛋白TgAtg8和虫株体内天然的TgAtg8蛋白。IFA实验表明,TgAtg8均匀分布于虫体胞浆中,但在自噬诱导培养后,TgAtg8逐渐聚集成颗粒状。结论制备的特异性抗TgAtg8多克隆抗体可用于检测虫体内TgAtg8蛋白在弓形虫自噬形成过程中的变化情况。
Objective To prepare and evaluate specific-TgAtg8 polyclonal antibody. Methods The known Sac- charomyces cerevisiae Atg protein sequences were used to identify Toxoplasma gondii homologous protein through bioin- formaties analysis. TgAtg8 eDNA was amplified and cloned into prokaryotic expression vector pGEX-6p-1. The constructed pGEX-6p-l-TgAtg8 was transformed into E. coli BL21 cells and induced with IFFG for expression. The expression product was analyzed through SDS-PAGE and Western blotting. The recombinant TgAtg8 protein with an N-terminal glutathione-S transferase tag was used to immunize rabbits and raise specific polyclonal antibody against TgAtg8. Subsequently, the an- tibody was applied for Western blotting and IFA assay. Results Recombinant expression plasmid of pGEX-6p-l-TgAtg8 was confirmed correct by restriction enzyme digestion and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg8 protein with the predicted molecular weight （Mr40000） was expressed highly in E. coli BL21. After immunization, the specific antibodies against TgAtg8 protein were produced. The anti-TgAtg8 polyclonal antibody re- acted specifically with TgAtg8 fusion protein or endogenous TgAtg8. Importantly, IFA assay determined that the TgAtg8 signal was generally distributed throughout the cytoplasm of the taehyzoites. However, the green fluorescence signal gath- ered into one or more green spots after induction of autophagy. Conclusion The specific polyelonal antibody against TgAtg8 could be used to observe the dynamics of autophagosome formation in T. gondii, which is useful tool to investi- gate the autophagie machinery in this parasite.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2014年第2期130-134,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
浙江省自然科学基金资助项目(No.LY12H19004)~~
