多头带绦虫蛋白激酶A催化亚基基因的克隆及原核表达

doi:10.16431/j.cnki.1671-7236.2017.10.005

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (10): 2858-2864.doi: 10.16431/j.cnki.1671-7236.2017.10.005

多头带绦虫蛋白激酶A催化亚基基因的克隆及原核表达

杨洋¹, 李文卉¹, 张念章¹, 李婷婷¹, 李立¹, 闫鸿斌¹, 贾万忠^1,2, 付宝权^1,2

1. 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, 兰州 730046;
2. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2017-03-08 出版日期:2017-10-20 发布日期:2017-10-20
通讯作者: 付宝权 E-mail:fubaoquan@163.com
作者简介:杨洋(1990-),女,云南水富人,硕士生,研究方向:人畜共患病及兽医公共卫生学,E-mail:yangyang2014418@163.com
基金资助:
甘肃省自然科学基金（1506RJZA152）

Cloning and Prokaryotic Expression of Protein Kinase A Catalytic Subunit Gene from Taenia multiceps

YANG Yang¹, LI Wen-hui¹, ZHANG Nian-zhang¹, LI Ting-ting¹, LI Li¹, YAN Hong-bin¹, JIA Wan-zhong^1,2, FU Bao-quan^1,2

1. Key Laboratory of Veterinary Parasitology of Gansu Province, Key Laboratory Veterinary Public Health of the Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Aminimal Infectious Disease, Yangzhou 225009, China

Received:2017-03-08 Online:2017-10-20 Published:2017-10-20

摘要/Abstract

摘要：

为鉴定多头带绦虫蛋白激酶A催化亚基（TmPKA-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TmPKA-C基因。将TmPKA-C基因片段连接至pET-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，TmPKA-C基因开放阅读框的长度为1 032 bp，可编码343个氨基酸。TmPKA-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。

关键词: 多头带绦虫; TmPKA-C; 原核表达; 反应原性

Abstract:

To study the potential of protein kinase A catalytic subunit of Taenia multiceps (TmPKA-C) gene as a diagnostic antigen, the TmPKA-C gene was amplified by RT-PCR from the total RNA of Taenia multiceps. The TmPKA-C gene fragment was ligated into prokaryotic expression vector pET-30a and transformed into Escherichia coli Transetta (DE3) strain, then the reactonogenicity of recombinant TmPKA-C protein was analyzed by Western blotting and indirect ELISA. The open reading frame of TmPKA-C gene was 1 032 bp, encoding 343 amino acids. The expression product of TmPKA-C gene mainly existed in the form of inclusion body, and the molecular weight of recombinant protein was about 42 ku. Western blotting analysis showed that the recombinant protein could react specifically with the sera from naturally infected sheep with coenurosis, and it could also have specific reaction with the sera of artificial infected sheep with coenurosis. The indirect ELISA analysis showed that the sera from the coenurosis of sheep could react specifically with the recombinant protein. These results proved that the recombinant protein had good reactionogenicity. This study laid a foundation for the search of new diagnostic antigens for coenurosis of sheep.

Key words: Taenia multiceps; TmPKA-C; prokaryotic expression; reactinogenicity

中图分类号:

S852.73⁺4

杨洋, 李文卉, 张念章, 李婷婷, 李立, 闫鸿斌, 贾万忠, 付宝权. 多头带绦虫蛋白激酶A催化亚基基因的克隆及原核表达[J]. 《中国畜牧兽医》, 2017, 44(10): 2858-2864.

YANG Yang, LI Wen-hui, ZHANG Nian-zhang, LI Ting-ting, LI Li, YAN Hong-bin, JIA Wan-zhong, FU Bao-quan. Cloning and Prokaryotic Expression of Protein Kinase A Catalytic Subunit Gene from Taenia multiceps[J]. , 2017, 44(10): 2858-2864.

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多头带绦虫蛋白激酶A催化亚基基因的克隆及原核表达

Cloning and Prokaryotic Expression of Protein Kinase A Catalytic Subunit Gene from Taenia multiceps

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