【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。更多还原

【Abstract】 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.更多还原