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

发布时间：2018-04-15 15:14

本文选题：旋毛虫肌幼虫 + 谷氨酰胺酶　；参考：《中国兽医科学》2017年05期

【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。
[Abstract]:The full-length Glutamase TsGls gene was amplified from the muscle larvae of Trichinella spiralis by RT-PCR and cloned into the expression vector pET-32a (). After sequencing, it was transformed into the host strain Rosetta(DE3)pLysS, and the recombinant protein TsGls was induced by IPTG.The polyclonal antibody was prepared by immunizing rabbits with purified TsGls protein and the TsGls in muscle larvae of Trichinella spiralis was detected by immunofluorescence.The results showed that the recombinant plasmid pET-32a (pET-32a) was successfully constructed, the molecular weight of the recombinant TsGls protein was about 72kuby SDS-PAGE analysis, and the titer of the polyclonal antibody was 1.024 脳 10 ~ (6) 渭 m Western-blot. The results showed that the polyclonal antibody was highly reactive to the recombinant TsGls.Immunofluorescence localization showed that TsGls protein existed in the subcutaneous tissue of Trichinella spiralis muscle larvae.The results laid a foundation for further study on glutamate-dependent acid-tolerant mechanism of Trichinella spiralis.
【作者单位】：东北农业大学动物医学学院;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;
【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)
【分类号】：S852.7

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