目录

为了表达牛环形泰勒虫（Theileria annulata）截短HSP70基因编码蛋白并研究该蛋白的结构与功能特性，本研究扩增牛环形泰勒虫HSP70目的基因并构建重组质粒pMD18-T-HSP70，选取其他种的同源HSP70蛋白序列构建系统进化树；利用生物信息学方法分析HSP70基因编码蛋白的氨基酸组成、基本理化性质、亲疏水性、跨膜区结构、信号肽、可能的磷酸化位点、亚细胞定位及蛋白的二级结构和三级结构；对重组蛋白HSP70进行蛋白互作网络分析；构建原核表达载体pET28a-HSP70，筛选诱导表达条件，镍柱纯化重组蛋白及检测反应原性。结果显示，牛环形泰勒虫HSP70蛋白序列与小泰勒虫的序列同源性较高，蛋白分子质量为42 ku，理论等电点（pI）为5.61，属于酸性亲水性蛋白，无跨膜区及信号肽；蛋白功能预测结果显示，HSP70包含32个可能的磷酸化位点，亚细胞定位分析显示该蛋白主要分布于细胞质。蛋白质二级结构中α-螺旋、β-转角、无规则卷曲、延伸链分别占39.18%、8.51%、30.41%和21.91%。蛋白互作网络构建结果显示，与HSP70相互作用的蛋白主要为HSP90家族成员，另外还有伴侣蛋白GrpE同系物，预示着HSP70可能在细胞内与HSP90形成复合体发挥作用。本试验成功构建原核表达载体，获得了大小约为48 ku的融合蛋白，以0.6 mmol/L IPTG于37 ℃诱导5 h，蛋白表达较好；点状印迹及Western blotting结果表明，表达产物可被自然感染的牛环形泰勒虫阳性血清识别，具有良好的反应原性。本试验结果为进一步探讨牛环形泰勒虫HSP70功能机制提供了理论依据。

Cloning,Expression,Characterization and Protein-protein Interaction Analysis of Thelieria annulata HSP70 Gene

To express and predict the structural and function characteristics of the protein encoded by the truncated HSP70 gene of Theileria annulata,this study was performed on the target gene HSP70,and the gene sequence was amplified,subsequently the recombinant plasmid pMD18-T-HSP70 was constructed.Phylogenetic tree was built with the homologous HSP70 protein sequences from other HSP70 protein sequences.Bioinformatics approaches were used to analyze HSP70 protein characteristics,which included amino acids composition,basic physicochemical properties,hydrophilicity/hydrophobicity,transmembrane structure,signal peptide,possible phosphorylation sites,subcellular localization,and secondary and tertiary structures of proteins.In addition,the protein-protein interaction (PPI) network analysis of the recombinant protein HSP70 was carried out.The prokaryotic expression vector pET28a-HSP70 was constructed,and the protein expression conditions were optimized.Purification of recombinant protein was performed by His-trap affinity chromatography,and reactogenicity was tested.The results showed that the sequence of HSP70 protein of Thelieria annulata had high homology with that of Theileria parva.The molecular weight of the protein was 42 ku,the theoretical isoelectric point (pI) was 5.61,it belonged to acidic protein and hydrophilic protein,without transmembrane domain and signal peptide.Protein function prediction results showed that HSP70 contained 32 possible phosphorylation sites,the sub-cellular location analysis showed that the protein was mainly distributed in the cytoplasm.The alpha helix,beta turn,random coil and extended chain accounted for 39.18%,8.51%,30.41% and 21.91%,respectively.PPI network construction results showed that the proteins interacting with HSP70 were mainly HSP90 family members,in addition to the chaperone protein GrpE homologue,which indicated that HSP70 might form a complex with HSP90 in the cell.The prokaryotic expression vector was successfully constructed,and obtained a infusion protein with 48 ku,the optimal expression condition was at 0.6 mmol/L IPTG induced at 37 ℃ for 5 h.The results of dot blotting and Western blotting showed that the expression product could be recognized by serum of the horses naturally infected by Thelieria annulata,and the reactogenicity was ideal.The results of this experiment would provide a theoretical basis for further exploration of the functional mechanism of Thelieria annulata HSP70.