Cloning and prokaryotic expression of Theileria equi rhoptry neck protein 5 gene

doi:10.12140/j.issn.1000-7423.2023.04.017

Abstract

Abstract:

The rhoptry-neck protein 5 (ron5) gene was PCR amplified from the Xinjiang isolates of Theileria equi. The prokaryotic expression vector of pGEX-4T-ron5 was constructed and transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The expression products was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting analysis was performed using glutathione S-transferase labeled rabbit monoclonal antibody (1:2 000) and horse serum infected with T. equi (1:100) as primary antibodies, and horseradish peroxidase labeled sheep anti-rabbit IgG and rabbit anti-horse IgG (1:5 000) as secondary antibodies. The ron5 of the Xinjiang isolate of T. equi was 1 434 bp, and the entry number obtained by GenBank was OP777492. Sequence comparison results showed that the nucleotide and amino acid sequences of ron5 of the Xinjiang isolate were the most consistent with those of T. equi WA strain (GenBank accession number XM 004833657), which were 99.6% and 99.2%, respectively. Phylogenetic tree analysis showed that the Xinjiang isolate and the T. equi WA strain were clustered on the same branch, with the most closely relation. SDS-PAGE analysis showed that the protein expression was the highest at 3 h induced by isopropyl thiogalactoside. The relative molecular mass (M_r) of RON5 is about 79 000, which is mainly expressed in the form of inclusion bodies. Western blotting results showed that RON5 could be recognized by glutathione S-transferase labeled antibody and horse serum infected with T. equi, with an obvious band at M_r79 000. In this study, ron5 was successfully cloned, and the RON5 expressed had good antigenicity.

Key words: Theileria equi, Rhoptry-neck protein 5, Prokaryotic expression, Antigenicity

CLC Number:

R382.9

LU Xing, WANG Shuiyi, CHEN Linjun, LIU Mingming, LIU Yutong, ZHU Huiru, JIANG Bingbing, DU Shaolei, BAYIN Chahan, LIU Dandan, ZHANG Wei. Cloning and prokaryotic expression of Theileria equi rhoptry neck protein 5 gene[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(4): 497-501.

Figures/Tables 3

References 22

[1]	Malekifard F, Tavassoli M, Yakhchali M, et al. Detection of Theileriaequi and Babesia caballi using microscopic and molecular Methods in horses in suburb of Urmia, Iran[J]. Vet Res Forum, 2014, 5(2): 129-133. pmid: 25568706
[2]	Luo J, Liu GY, Tian ZC, et al. Cloning and phylogenetic analysis of EMA1 gene of Theileria equi[J]. Chin Vet Sci, 2012, 42(4): 352-356. (in Chinese)
	(罗金, 刘光远, 田占成, 等. 马泰勒虫EMA1基因的克隆与生物学特性分析[J]. 中国兽医科学, 2012, 42(4): 352-356.)
[3]	Jiang JS. Animal protozoology[M]. Beijing: China Agricultural University Press, 2000: 7. (in Chinese)
	(蒋金书. 动物原虫病学[M]. 北京: 中国农业大学出版社, 2000: 7.)
[4]	Yang HX, Zhang Y, Liu SF, et al. Report on pregnant horses killed by horse Taylor disease[J]. Chin J Vet Med, 2017, 53(11): 50-51. (in Chinese)
	(杨红霞, 张杨, 刘世芳, 等. 马泰勒虫病致死孕马报道[J]. 中国兽医杂志, 2017, 53(11): 50-51.)
[5]	Liu Z, Zhang YW, Li XX, et al. An overview of equine piroplasmosis and its diagnostic methods[J]. Chin Anim Health Insp, 2019, 36(10): 66-70. (in Chinese)
	(刘铮, 张义伟, 李茜茜, 等. 马梨形虫病及其诊断方法概述[J]. 中国动物检疫, 2019, 36(10): 66-70.)
[6]	Mital J, Meissner M, Soldati D, et al. Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion[J]. Mol Biol Cell, 2005, 16(9): 4341-4349. doi: 10.1091/mbc.e05-04-0281
[7]	Xu JL, Wang JM, Liu JL, et al. Cloning and characterization of the rhoptry neck protein 2 gene in ovine Babesia species in China[J]. Chin Vet Sci, 2019, 49(5): 551-559. (in Chinese)
	(徐建林, 王锦明, 刘军龙, 等. 我国羊巴贝斯虫棒状体颈部蛋白RON2基因的克隆与结构和遗传进化分析[J]. 中国兽医科学, 2019, 49(5): 551-559.)
[8]	Qi NS. Cloning and expressing profiles of Eimeria tenella rhoptry neck protein 2 (EtRON2) gene during the different development stages[D]. Nanjing: Nanjing Agricultural University, 2010: 2-7. (in Chinese)
	(戚南山. 柔嫩艾美耳球虫棒状体颈部蛋白2(EtRON2)基因的克隆表达及功能初步研究[D]. 南京: 南京农业大学, 2010: 2-7.)
[9]	Beck JR, Chen AL, Kim EW, et al. RON5 is critical for organization and function of the Toxoplasma moving junction complex[J]. PLoS Pathog, 2014, 10(3): e1004025. doi: 10.1371/journal.ppat.1004025
[10]	Sun YY, Liao SQ, Li YL, et al. Research progress of rhoptry proteins as apicomplexan vaccine candidates[J]. Chin J Anim Infect Dis, 2022, 30(1): 203-210. (in Chinese)
	(孙芸芸, 廖申权, 李跃龙, 等. 顶复门原虫棒状体蛋白疫苗候选分子的研究进展[J]. 中国动物传染病学报, 2022, 30(1): 203-210.)
[11]	Alexander DL, Mital J, Ward GE, et al. Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles[J]. PLoS Pathog, 2005, 1(2): e17. doi: 10.1371/journal.ppat.0010017 pmid: 16244709
[12]	Straub KW, Cheng SJ, Sohn CS, et al. Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements[J]. Cell Microbiol, 2009, 11(4): 590-603. doi: 10.1111/j.1462-5822.2008.01276.x pmid: 19134112
[13]	Nozaki M, Baba M, Tachibana M, et al. Detection of the rhoptry neck protein complex in Plasmodium sporozoites and its contribution to sporozoite invasion of salivary glands[J]. mSphere, 2020, 5(4): e00325-e00320.
[14]	Curtidor H, Patiño LC, Arévalo-Pinzón G, et al. Plasmodium falciparum rhoptry neck protein 5 peptides bind to human red blood cells and inhibit parasite invasion[J]. Peptides, 2014, 53: 210-217. doi: 10.1016/j.peptides.2013.07.028
[15]	Wang M, Cao SN, Du NL, et al. The moving junction protein RON4, although not critical, facilitates host cell invasion and stabilizes MJ members[J]. Parasitology, 2017, 144(11): 1490-1497. doi: 10.1017/S0031182017000968 pmid: 28662729
[16]	Wang M. Functional study on moving junction complex member rhoptry neck protein 4 of Toxoplamsa gondii[D]. Harbin:Northeast Agricultural University, 2017: 9-11. (in Chinese)
	(王铭. 弓形虫移动联结复合物成员棒状体颈部蛋白4的功能研究[D]. 哈尔滨: 东北农业大学, 2017: 9-11.)
[17]	Fan SL, Liu DD, Wei LT, et al. Prokaryotic expression and identification of apical membrane antigen 1 gene of Theileria equi[J]. Chin J Vet Sci, 2021, 41(9): 1752-1756, 1769. (in Chinese)
	(范士龙, 刘丹丹, 韦丽婷, 等. 马泰勒虫顶膜抗原1基因的原核表达与鉴定[J]. 中国兽医学报, 2021, 41(9): 1752-1756,1769.)
[18]	Xiao PP, Zhang MY, Telieke K, et al. Prokaryotic expression of RAP-1 gene of Theileria equi and characteristic analysis on the encoded protein[J]. Chin Anim Health Insp, 2020, 37(7): 104-111. (in Chinese)
	(肖培培, 张梦圆, 特列克•库拉别克, 等. 马泰勒虫RAP-1基因的原核表达及蛋白性质分析[J]. 中国动物检疫, 2020, 37(7): 104-111.)
[19]	Zhang W, Fan SL, Wei LT, et al. Prokaryotic expression and bioinformatics analysis of RON2 gene in Theileria equi[J]. J Northwest A F Univ Nat Sci Ed, 2021, 49(12): 18-27. (in Chinese)
	(张伟, 范士龙, 韦丽婷, 等. 马泰勒虫RON2基因的原核表达及生物信息学分析[J]. 西北农林科技大学学报(自然科学版), 2021, 49(12): 18-27.)
[20]	Ngabu M. The proteins of moving junction of Babesia orientalis RON2, RON4 and RON5: identification and analysis[D]. Wuhan: Huazhong Agricultural University, 2017: 59-64.
[21]	Song RQ, Huercha, Zhai XJ, et al. Prokaryotic expression of the cysteine proteinase gene of Xinjiang strain of Theileriaequi/Babesia caballi and its bioinformatics analysis[J]. Chin J Anim Vet Sci, 2020, 51(10): 2547-2556. (in Chinese)
	(宋瑞其, 呼尔查, 翟雪洁, 等. 马泰勒虫和驽巴贝斯虫半胱氨酸蛋白酶截短基因的克隆表达及其生物信息学分析[J]. 畜牧兽医学报, 2020, 51(10): 2547-2556.)
[22]	Zhao Y, Li ZY, Chen J, et al. Protective efficacy of pVAX-RON5p against acute and chronic infections of Toxoplasma gondii in BALB/c mice[J]. Exp Parasitol, 2016, 163: 24-30. doi: 10.1016/j.exppara.2016.01.011 pmid: 26821295