摘要
【目的】获得牛环形泰勒虫(Theileria annulata)新疆株enolase基因,并分析其生物学特性及反应原性。【方法】对牛环形泰勒虫enolase基因进行扩增和克隆,构建原核表达载体pGEX-4T-1-enolase,诱导表达enolase重组蛋白并进行蛋白纯化,通过Western blotting验证enolase重组蛋白反应原性。利用生物信息学方法对enolase基因编码蛋白的理化性质、亲疏水性、跨膜区、信号肽、磷酸化、亚细胞定位及蛋白互作网络进行预测分析。【结果】PCR扩增出大小为1248 bp的牛环形泰勒虫enolase基因片段,enolase重组蛋白大小约70 ku;Western blotting结果表明,该重组蛋白与牛环形泰勒虫阳性血清发生反应。enolase基因编码416个氨基酸,理论等电点为5.91,有38个磷酸化位点;二级结构主要由α-螺旋(43.03%)和无规则卷曲(33.17%)组成;具有17个B细胞抗原表位,亚细胞定位主要位于细胞质中;enolase蛋白与磷酸甘油酸变位酶(PGAM)、二磷酸核苷激酶(NDK)、磷酸甘油酸激酶(PGK)、热休克蛋白70(HSP70)存在相互作用。【结论】本试验成功克隆出新疆牛环形泰勒虫enolase基因,蛋白互作网络预测其与糖酵解和能量代谢相关的蛋白相互作用。研究结果为牛环形泰勒虫能量代谢途径基因的相关研究奠定基础。
【Objective】 This study was aimed to obtain enolase gene from Xinjiang strain of Theileria annulata(T. annulata),and analyze the biological characteristics and reactionogenicity of the protein.【Method】 The enolase gene of T.annulata was amplified and cloned, and the prokaryotic expression vector pGEX-4 T-1-enolase was constructed.The recombinant enolase protein was induced and purified.The reactivity of the recombinant enolase protein was verified by Western blotting.Bioinformatics methods were used to predict the physicochemical properties, hydrophilicity and hydrophobicity, transmembrane domain, signal peptide, phosphorylation, subcellular localization and protein interaction network of enolase gene encoded protein.【Result】 A 1 248 bp enolase gene fragment was amplified by PCR.The size of enolase recombinant protein was about 70 ku.The results of Western blotting showed that the recombinant protein reacted with the positive serum of T.annulata.enolase gene encoded 416 amino acids with a theoretical isoelectric point of 5.91 and 38 phosphorylation sites.The secondary structure was mainly composed of alpha helix(43.03%) and random coil(33.17%).It had 17 B cell epitopes, and the subcellular localization was mainly located in the cytoplasm.enolase protein interacted with phosphoglycerate mutase(PGAM),nucleoside diphosphate kinase(NDK),phosphoglycerate kinase(PGK) and heat shock protein 70(HSP70).【Conclusion】 In this study, the enolase gene of T. annulata from Xinjiang was successfully cloned. The protein-protein interaction network predicted the protein interaction of enolase gene with glycolysis and energy metabolism. The results of this study laid a foundation for the related research on the energy metabolism pathway genes of T. annulata.
作者
郑会珍
普浩
缪荣浩
甘露
葛晓敏
巴音查汗
李永畅
郭庆勇
ZHENG Huizhen;PU Hao;MIAO Ronghao;GAN Lu;GE Xiaomin;Bayinchahan;LI Yongchang;GUO Qingyong(College of Veterinary Medicine,Xingjiang Agricultural University,Urumqi 830052,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第10期3972-3981,共10页
China Animal Husbandry & Veterinary Medicine
基金
新疆维吾尔自治区自然科学项目“新疆牛绵羊无浆体和牛环形泰勒虫ELISA试剂盒的建立及应用”(XJEDU2020I008)
新疆农业大学博士后项目
2021年“天池博士计划”。
作者简介
郑会珍,联系方式:E-mail:1974741550@qq.com。;通信作者:李永畅,E-mail:lyc_8762017@163.com;通信作者:郭庆勇,E-mail:wlmqgqy@126.com。
