肌旋毛虫谷氨酰胺酶基因的原核表达及其免疫荧光定位研究  

Prokaryotic expression and immunofluorescent localization of glutaminase gene in Trichinella spiralis muscle larvae

作　　者：赵祥[1,2] 于洁[1,2] 胡静[1,2] 许静秀 韩彩霞[1,2] 李晓云[1,2] 宋铭忻[1,2] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2017年第5期587-591,共5页Chinese Veterinary Science

基　　金：家畜疫病病原生物学国家重点实验室开放课题(SKLVEB2015KFKT010);国家自然科学基金项目(31172312)

摘　　要：采用RT-PCR方法从旋毛虫肌幼虫中扩增出谷氨酰胺酶(TsGls)基因全长序列,将其克隆到表达载体pET-32a(+)中,测序验证后转化到表达宿主菌Rosetta(DE3)pLysS中,用IPTG诱导表达重组蛋白TsGls。以纯化的TsGls蛋白免疫家兔制备多克隆抗体,并对旋毛虫肌幼虫中的TsGls进行免疫荧光定位。结果显示,成功构建了重组质粒pET-32a(+)-TsGls;经SDS-PAGE分析,重组TsGls蛋白的分子质量约为72 ku;制备的多克隆抗体效价可达1.024×10~6;Western-blot分析结果显示,多克隆抗体与重组TsGls具有较强反应性;免疫荧光定位显示,TsGls蛋白存在于旋毛虫肌幼虫皮下组织。上述研究结果为进一步研究旋毛虫谷氨酸依赖型抗酸机制奠定了基础。To analyze the enzymatic immunogenicity and reactivity of recombinant glutaminese decarboxylase TsGls protein of Trichinellaspiralis muscle larvae,the complete sequence of TsGls gene was amplified by RT-PCR,cloned into prokaryotic expression plasmid pET-32a（＋）,transformed into Ros- ette （DE3）,and then induced with IPTG. The polyclonal antibodies against the recombinant protein was prepared with the purified protein and then the immunofluorescent localization of TsGls in Trichinella spiralis muscle larvae was studied. The recombinant plasmid pET-32a（4-）-TsGls was successfully const- ructed. The recombinant protein rTsGls was confirmed to be about 72 ku in size by SDS-PAGE. The high polyclonal antibody titer was up to 1.024 X 106, indicating good immunogenicity of rTsGls. Western-blot analysis showed high reactivity between rTsGls and larval extracts. Immunofluorescent showed that Ts- Gls protein existed in the subcutaneous tissue of Trichinella spiralis. The above-mentioned results laid foundation for further study of glutamate-dependent acid mechanisms of rrichinella spiralis.

关 键 词：旋毛虫肌幼虫 谷氨酰胺酶 原核表达 免疫荧光定位 

分 类 号：S855.9[农业科学—临床兽医学]