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

摘要/Abstract

摘要： 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。

关键词: 牛环形泰勒虫; HSP70基因; 原核表达; 反应原性; 生物信息学分析

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

中图分类号:

樊新丽, 宋瑞其, 呼尔查, 李敏, 翟雪洁, 郝蕴伟, 巴音查汗, 张杨. 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[J]. 中国畜牧兽医, 2021, 48(1): 1-11.

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.

参考文献

[1] 马全英,李有全,刘军龙,等.环形泰勒虫喀什株感染牛淋巴细胞系的建立及其生物学特征的鉴定[J].中国兽医科学,2019,49(2):144-151. MA Q Y,LI Y Q,LIU J L,et al.Establishment and characterization in vitro culture of a Theileria annulata Kashi-infected bovine lymphocyte cell lines[J].Chinese Veterinary Science,2019,49(2):144-151.(in Chinese)
[2] 相权珈,关贵全,刘爱红,等.环形泰勒虫SPAG蛋白的原核表达及反应原性分析[J].中国兽医科学,2020,50(3):269-275. XIANG Q J,GUAN G Q,LIU A H,et al.Prokaryotic expression and reactiongenicity analysis of recom-binant Theileria annulata SPAG protein[J].Chinese Veterinary Science,2020,50(3):269-275.(in Chinese)
[3] ALTAY K,AKTAS M.Molecular detection of Thelieria and Babesia infections in cattle[J].Veterinary Parasitology,2008,158(4):295-301.
[4] 李佳,段真真,杨世忠,等.阿克苏地区牛环形泰勒虫病分子流行病学调查[J].现代农业科技,2019,19:219-223. LI J,DUAN Z Z,YANG S Z,et al.Molecular epidemiology investigation of Thelieria annulata in Aksu region[J].Modern Agricultural Technology,2019,19:219-223.(in Chinese)
[5] HUNT C,MORIMOTO R I.Conserved features of eukaryotic HSP70 genes revealed by comparison with the nucleotide sequence of human HSP70[J].Proceeding of the National Academy of Science of the United States of America,1985,82(19):6455-6459.
[6] KAMPINGA H H,HANGEMAN J,VOS M J,et al.Guidelines for the nomenclature of the human heat shock proteins[J].Cell Stress Chaperones,2009,14(1):105-111.
[7] LINDQUIST S.The heat-shock response[J].Annual Review Biochemistry,1986,55:1151-1191.
[8] RAFATI S,GHOLAMI E,HASSANI N,et al.Leishmania major heat shock protein 70(HSP70) is not protective in murine models of cutaneous leishmaniasis and stimulates strong humoral responses in cutaneous and visceral leishmaniasis patients[J].Vaccine,2007,25(21):4159-4169.
[9] 杨佳栋,刘月琴,张英杰,等.山羊HSPA6蛋白的特性分析及互作蛋白网络构建[J].中国畜牧兽医,2020,47(3):706-713. YANG J D,LIU Y Q,ZHANG Y J,et al.Characterization analysis of HSPA6 protein in goat and construction of interactive protein network[J].China Animal Husbandry & Veterinary Medicine,2020,47(3):706-713.(in Chinese)
[10] 蔺红玲,张继瑜,魏小娟,等.环形泰勒虫主要膜蛋白的研究进展[J].黑龙江畜牧兽医,2011,5:22-24. LIN H L,ZHANG J Y,WEI X J,et al.Research progress on the main membrane proteins of Theileria annulata[J].Heilongjiang Animal Science and Veterinary Medicine,2011,5:22-24.(in Chinese)
[11] ZUIDERWEG E R,HIGHTWER L E,GESTWICKI J E.The remarkable multivalency of the Hsp70 chaperones[J].Cell Stress Chaperones,2017,22(2):173-189.
[12] RITOSSA F M.A Bew puffing pattern induced by heat shock and DNP in Drosophila[J].Cellular and Molecular Life Sciences,1962,18(12):571-573.
[13] KREGEL K C.Heat shock protein:Modifying factors in physiological stress responses and acquired thermotolerance[J].Journal of Applied Physiology,2002,92:2177-2186.
[14] MILLAR N L,MURRELL G A C.Heat shock proteins in tendinopathy:Novel molecular regulatoras[J].Mediators Inflammation,2012,2012:436203.
[15] 郭会灿.蛋白质翻译后修饰研究进展[J].生物技术通报,2011,7:18-21. GUO H C.Research progress of protein translational modifications[J].Biotechnology Bulletin,2011,7:18-21.(in Chinese)
[16] 戴忠良,陈丽,山溪,等.甘蓝晚抽薹基因BoFLC3克隆、序列分析和亚细胞定位[J].江苏农业学报,2018,34(6):1324-1330. DAI Z L,CHEN L,SHAN X,et al.Cloning,sequence analysis and subcellular localization of Brassica oleracea BoFLC3 gene[J].Jiangsu Journal of Agricultural Sciences,2018,34(6):1324-1330.(in Chinese)
[17] VELASCO L,DUBLANG L,MORO F,et al.The complex phosphorylation patterns that regulate the activity of HSP70 and its cochaperones[J].International Journal of Molecular Sciences,2019,20(17):4122-4131.
[18] PACI V,KRASTEVA I,ORSINI M,et al.Proteomic analysis of Brucella melitensis and Brucella ovis for identification of virulence factor using bioinformatics approachs[J].Molecular and Cellular Probes,2020,2020:101581.
[19] 周剑,何深一,刘晓雨,等.弓形虫ROP31基因克隆及生物信息学分析[J].中国病原生物学杂志,2018,13(5):462-467. ZHOU J,HE S Y,LIU X Y,et al.Cloning and bioinformatics analysis of ROP31 in Toxoplasma gondii[J].Journal of Pathogen Biology,2018,13(5):462-467.(in Chinese)
[20] RADEMACHER T,SACK M,ARCALIS E,et al.Recombinnant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominanatly single-GlcNAc N-glycans[J].Plant Biotechnology Journal,2008,6(2):189-201.
[21] NITIKA N,TRUMAN A W.Cracking the chaperone code:Cellular roles for HSP70 phosphorylation[J].Trends in Biochemical Sciences,2017,42(12):932-935.
[22] ALVIRA S,CUELLAR J,RHL A,et al.Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop[J].Nature Communication,2014,5:5484.