牛环形泰勒虫HSP70基因的克隆表达、特性分析及互作蛋白网络构建

doi:10.16431/j.cnki.1671-7236.2021.01.001

Abstract

Abstract: 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.

Key words: Theileria annulata; HSP70 gene; prokaryotic expression; reactogenicity; bioinformatics analysis

CLC Number:

FAN Xinli, SONG Ruiqi, HU Ercha, LI Min, ZHAI Xuejie, HAO Yunwei, Bayinchahan, ZHANG Yang. Cloning,Expression,Characterization and Protein-protein Interaction Analysis of Thelieria annulata HSP70 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 1-11.

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

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