多头带绦虫蛋白激酶A催化亚基基因的克隆及原核表达

发布时间：2018-07-31 17:42

【摘要】：为鉴定多头带绦虫蛋白激酶A催化亚基(Tm PKA-C)基因作为诊断抗原的潜能,本试验以多头带绦虫成虫RNA为模板,利用RT-PCR扩增Tm PKA-C基因。将Tm PKA-C基因片段连接至p ET-30a原核表达载体进行原核表达,然后利用Western blotting和间接ELISA分析该蛋白的反应原性。结果表明,Tm PKA-C基因开放阅读框的长度为1 032 bp,可编码343个氨基酸。Tm PKA-C基因的表达产物主要以包涵体的形式存在,重组蛋白的分子质量大小约42 ku。Western blotting结果表明,该重组蛋白既能与自然感染脑多头蚴的绵羊血清发生特异性反应,又能与人工感染脑多头蚴不同时间段的绵羊血清发生特异性反应,表明该重组蛋白具有良好的反应原性。间接ELISA分析结果表明,脑多头蚴绵羊血清能与重组蛋白发生特异性反应,进一步证明该重组蛋白的反应原性良好。本研究为筛选脑多头蚴病诊断新抗原奠定了基础。
[Abstract]:In order to identify the potential of protein kinase A catalytic subunit (TM PKA-C) gene of Taenia multiceps as diagnostic antigen, the TM PKA-C gene was amplified by RT-PCR using RNA of Taenia multiceps as template. The TM PKA-C gene fragment was ligated into the prokaryotic expression vector of p ET-30a for prokaryotic expression, and the reactivity of the protein was analyzed by Western blotting and indirect ELISA. The results showed that the length of the open reading frame of Tm PKA-C gene was 1 032 BP, and the expression products of the gene encoding 343 amino acids. TM PKA-C mainly existed in the form of inclusion bodies. The molecular weight of the recombinant protein was about 42 ku.Western blotting. The recombinant protein can react with both sheep serum infected naturally and sheep sera infected with cerebrum polycaria at different time points, which indicates that the recombinant protein has good reactivity. The results of indirect ELISA analysis showed that the specific reaction between the recombinant protein and the serum of cerebral polycaria sheep was specific, which further proved that the reactivity of the recombinant protein was good. This study laid a foundation for screening new antigens for the diagnosis of cerebral polycephalosis.
【作者单位】：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农业部兽医公共卫生重点开放实验室甘肃省动物寄生虫病重点实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;
【基金】：甘肃省自然科学基金(1506RJZA152)
【分类号】：S852.734

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