猪带绦虫cAMP依赖性蛋白激酶催化亚基(PKA-C)基因的鉴定及表达  

Identification and analysis of expression of cAMP-dependent protein kinase catalytic subunit gene from Taenia solium

作　　者：刘光学[1] 王力[1] 郭爱疆[1,2] 张少华[1] 郑亚东[1,2] 侯俊玲[1] 骆学农[1] 

机构地区：[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实验室甘肃省动物寄生虫病重点实验室,甘肃兰州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,江苏扬州225009

出　　处：《中国兽医科学》2016年第6期671-677,共7页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31372433)

摘　　要：为鉴定猪带绦虫cAMP依赖性蛋白激酶催化亚基基因(Ts PKA-C),分析其作为诊断抗原的可行性,利用猪带绦虫基因组和转录组数据设计猪带绦虫Ts PKA-C特异引物,以猪带绦虫成虫总RNA为模板,通过RT-PCR扩增Ts PKA-C基因的完整ORF,并通过生物信息学分析进行鉴定。构建p ET-30a(+)-Ts PKA-C重组表达载体,在大肠杆菌BL21(DE3)中进行诱导表达。纯化的目的蛋白进行SDS-PAGE和Western-blot分析。结果表明,Ts PKA-C的ORF由1 032个核苷酸组成,编码343个氨基酸残基,与细粒棘球蚴和多房棘球蚴氨基酸序列的一致性分别达78.7%和98.0%;Ts PKA-C具有蛋白激酶A的结构域及其催化亚基特征性活性位点,且存在9个潜在的线性B淋巴细胞抗原表位。Ts PKA-C基因的表达产物主要以包涵体形式存在,且可与猪囊尾蚴病阳性血清发生反应,在42 ku处产生特异条带。本研究克隆并鉴定了Ts PKA-C基因,其表达产物可被猪囊尾蚴病阳性血清所识别。To identify c AMP-dependent protein kinase catalytic subunit（PKA-C） from Taenia solium and explore the probability to Ts PKA-C as a diagnostic antigen,the full-length sequence encoding Ts PKA-C gene was amplified from T.solium total RNA by RT-PCR and identified by bioinformatics.The fragment included Ts PKA-C complete ORF was cloned into the prokaryotic expression vector p ET-30a（＋）.The Ts PKA-C fusion protein was expressed in Escherichia coli and the immune-reactivity was tested by Western-blotting. The results indicated that Ts PKA-C gene sequence contained an ORF of 1 032 bp and the mature protein consisted of 343 amino acids. The sequence of amino acids of Ts PKA-C subunit was shared78.7% and 98.0%identity with Echinococcus granulosus and Echinococcus multilocularis,respectively.Multiple sequences alignment of the Ts PKA-C amino acid sequence with other important known parasitic helminth PKA-C from Gen Bank indicated that Ts PKA-C has PKA domains and conservative motifs.A total of 9linear B cell epitopes were predicted in Ts PKA-C.Western-blotting detected a specific band in 42 ku with the positive serum from porcine cysticercosis.The results from bioinformatics analysis and Westernblotting indicated that the fragment Ts PKA-C was cloned and its product can be recognized by the positive serum from porcine cysticercosis.

关 键 词：猪带绦虫 cAMP依赖性蛋白激酶催化亚基 鉴定 表达 诊断候选抗原 

分 类 号：S852.734[农业科学—基础兽医学]