毒害艾美耳球虫谷胱甘肽过氧化物酶EnGPX的原核表达与分析

doi:10.11843/j.issn.0366-6964.2024.02.040

摘要/Abstract

摘要： 旨在研究毒害艾美耳球虫谷胱甘肽过氧化物酶EnGPX的反应原性及其在虫体内的亚细胞定位。提取毒害艾美耳球虫(扬州株)配子体总RNA,RT-PCR扩增EnGPX的ORF编码序列,构建原核表达质粒pET-28a(+)-EnGPX,转化至BL21(DE3)进行体外诱导表达,同时制备鼠抗rEnGPX多克隆抗体,对重组蛋白进行Western blot反应原性分析和激光共聚焦免疫荧光定位分析。结果表明,EnGPX ORF序列全长753 bp,编码250个氨基酸,体外重组表达蛋白大小约30 ku,主要以包涵体形式存在。该重组蛋白能被6×HIS标签单克隆抗体,鼠抗rEnGPX多克隆抗体,毒害艾美耳球虫、巨型艾美耳球虫和柔嫩艾美耳球虫病鸡康复血清所识别,表明其具有较好的反应原性和交叉反应原性。在天然配子体蛋白中检测出EnGPX,其编码蛋白主要分布于配子体内的Ⅱ型成壁体(WFBII)及卵囊壁上。本研究成功克隆表达了毒害艾美耳球虫谷胱甘肽过氧化物酶EnGPX,验证其具有良好的反应原性,并定位于配子体及卵囊壁上。以期为研究EnGPX参与卵囊壁形成的分子机制提供新的线索,也为研制新型球虫亚单位疫苗提供新的靶标。

关键词: 毒害艾美耳球虫, EnGPX, 克隆表达, 反应原性, 免疫荧光定位

Abstract: The aim of this paper was to study the antigenicity of glutathione peroxidase EnGPX and its subcellular localization in Eimeria necatrix. Total RNA was extracted from the gametophyte of E. necatrix (Yangzhou strain), and the ORF coding sequence of EnGPX was amplified using RT-PCR. The prokaryotic expression plasmid pET-28a(+)-EnGPX was constructed and transformed into BL21(DE3) for in vitro expression, additionally, a mouse anti-rEnGPX polyclonal antibody was prepared and used to analyze the recombinant protein through Western blotting, and laser confocal immunofluorescence localization analysis. The study found that the coding region of the EnGPX gene was 753 base pairs in length and encoded a protein consisting of 250 amino acids. The resulting recombinant protein had a molecular weight of around 30 ku and was predominantly present in the form of inclusion bodies. The recombinant protein exhibited favorable reactivity and cross-reactivity, as it was recognized by a 6×HIS-tagged monoclonal antibody, a mouse anti-rEnGPX polyclonal antibody, and convalescent serum from E. necatrix, E. maxima, and E. tenella. EnGPX was detected in natural gametophyte proteins, with the encoded protein primarily localized to the type II wall-forming body (WFBII) of the gametophyte and oocyst wall. This study successfully cloned and expressed the glutathione peroxidase (EnGPX) of E. necatrix. The recombinant protein exhibited good reactivity, and natural EnGPX protein was found to be localized on the gametophyte and oocyst wall. The results shed light on the molecular mechanism of EnGPX involvement in oocyst wall formation and identify potential targets for the development of novel subunit vaccines against coccidia.

Key words: Eimeria necatrix, EnGPX, cloning and expression, reactivity, immunofluorescence localization

中图分类号:

彭月梅, 叶状, 汪飞燕, 王礼跃, 冯永翠, 王乐乐, 候照峰, 许金俊, 陶建平, 刘丹丹. 毒害艾美耳球虫谷胱甘肽过氧化物酶EnGPX的原核表达与分析[J]. 畜牧兽医学报, 2024, 55(2): 846-853.

PENG Yuemei, YE Zhuang, WANG Feiyan, WANG Liyue, FENG Yongcui, WANG Lele, HOU Zhaofeng, XU Jinjun, TAO Jianping, LIU Dandan. Procaryotic Expression and Analysis of the EnGPX of Eimeria necatrix[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 846-853.

参考文献

[1] LI H H, SUN L Y, JIANG Y Y, et al. Identification and characterization of Eimeria tenella EtTrx1 protein[J]. Vet Parasitol, 2022, 310:109785.
[2] OSHO S O, ADEOLA O. Impact of dietary chitosan oligosaccharide and its effects on coccidia challenge in broiler chickens[J]. Brit Poultry Sci 2019, 60(6):766-776.
[3] METWALY M S, DKHIL M A, AL-QURAISHY S. The potential role of Phoenix dactylifera on Eimeria papillata-induced infection in mice[J]. Parasitol Resh, 2012, 111(2):681-687.
[4] DKHIL M A, DELIC D, AL-QURAISHY S. Goblet cells and mucin related gene expression in mice infected with Eimeria papillata[J]. Scientific World Journal, 2013, 2013:439865.
[5] SUBRAMANIAN B M, SRIRAMAN R, RAO N H, et al. Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds[J]. Vaccine, 2008, 26:3489-3496.
[6] LILLEHOJ H S, DING X, DALLOUL R A, et al. Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants[J]. J Parasitol, 2005; 91(3):666-673.
[7] WITCOMBE D M, FERGUSON DAVID J P, BELLI S I, et al. Eimeria maxima TRAP family protein EmTFP250:subcellular localisation and induction of immune responses by immunisation with a recombinant C-terminal derivative[J]. Int J Parasitol, 2004, 34:861-872.
[8] BELLI S I, MAI K, SKENE C D, et al. Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima[J]. Vaccine, 2004, 22:4316-4325.
[9] LIU D D, WANG F Y, CAO L Q, et al. Identification and characterization of a cDNA encoding a gametocyte-specific protein of the avian coccidial parasite Eimeria necatrix[J]. Mol Biochem Parasitol, 2020, 240:111318.
[10] FAN X C, LIU T L, WANG Y, et al. Genome-wide analysis of differentially expressed profiles of mRNAs, lncRNAs and circRNAs in chickens during Eimeria necatrix infection[J]. Parasit Vector, 2020, 13:167.
[11] WILLIAMS R B. Anticoccidial vaccines for broiler chickens:pathways to success[J]. Avian Pathol, 2002, 31:317-53.
[12] 刘丹丹. 毒害艾美耳球虫配子体抗原基因的克隆表达与功能研究[D]. 扬州大学,2014.
LIU D D. Cloning, expression and function research of gametocyte antigen genes of Eimeria necatrix[D]. Yangzhou University, 2014.(in Chinese)
[13] POSSENTI A, CHERCHI S, BERTUCCINI L, et al. Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation[J]. Int J Parasitol, 2010, 40(14):1639-1649.
[14] MAI K, SHARMAN P A, WALKER R A, et al. Oocyst wall formation and composition in coccidian parasites[J]. Mem I Oswaldo Cruz, 2009, 104(2):281-289.
[15] 叶状,王乐乐,汪飞燕,等. 毒害艾美耳球虫氧化还原酶EnOXIO1的原核表达与定位分析[J]. 畜牧兽医学报,2022,53(05):1553-1561.
YE Z, WANG L L, WANG F Y, et al. Procaryotic expression and immunolocalization analysis of the EnOXI01 of Eimeria necatrix[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(05):1553-1561.(in Chinese)
[16] BELLI S I, SMITH N C, FERGUSON D J P. The coccidian oocyst:a tough nut to crack[J]. Trends Parasitol, 2006, 22(9):416-423.
[17] DEPONTE M. Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes[J]. BBA-Gen Subjects, 2013, 1830(5):3217-3266.
[18] BELLI S I, WALLACH M G, LUXFORD C, et al. Roles of tyrosine-rich precursor glycoproteins and dityrosine-and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima[J]. Eukaryot Cell, 2003, 2(3):456-464.
[19] SHARMAN P A. Identification of enzymes potentially involved in the formation of the oocyst wall of coccidian parasites[D]. Townsville:James Cook University, 2013.
[20] MAI K, SMITH N C, FENG Z P, et al. Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima[J]. Int J Parasitol, 2011, 41(11):1157-1164.
[21] FRITZ H M, BOWYER P W, BOGYO MATTHEW, et al. Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[J]. PLoS One, 2012, 7:e29955.
[22] TEMPLETON T J, LANCTO C A, VIGDOROVICH V, et al. The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma[J]. Infect Immun, 2004; 72(2):980-987.
[23] 刘丹丹,王乐乐,汪飞燕,等. 毒害艾美耳球虫成壁体参与卵囊壁形成机制的探析[M]//中国畜牧兽医学会兽医寄生虫学分会第一届青年科学家学术论坛.武汉:2019:34.
LIU D D, WANG L L, WANG, F Y, et al. Exploring the mechanism of Eimeria necatrix involved in oocyst wall formation[M]//The first Academic Forum for young scientists of Veterinary Parasitology Branch of Chinese Society of Animal Husbandry and Veterinary Medicine, Wuhan:2019:34.(in Chinese)
[24] ASGHARI A, MAJIDIANI H, NEMATI T, et al. Toxoplasma gondii tyrosine-rich oocyst wall protein:a closer look through an in silico prism[J]. Bio Res Int, 2021, 2021:1315618.
[25] 王艳伟. 谷胱甘肽过氧化物酶突变体的修饰及药学性质研究[D]. 长春:吉林大学,2022.
WANG Y W. Research on modification and pharmaceutical properties of glutathione peroxidase mutants[D]. Changchun:Jilin university, 2022.(in Chinese)
[26] DEAR T N, CAMPBELL K, RABBITTS T H. Molecular cloning of putative odorant-binding and odorant-metabolizing proteins[J]. Biochemistry, 1991, 30(43):10376-10382.
[27] FLOHÉ L, TOPPO S, ORIAN L. The glutathione peroxidase family:Discoveries and mechanism[J]. Free Radical Bio Med, 2022, 187:113-122.
[28] WANG L, ZHANG L, NIU Y, et al. Glutathione peroxidase 7 utilizes hydrogen peroxide generated by Ero1α to promote oxidative protein folding[J]. AntiRedox Sign, 2014, 20(4):545-556.
[29] RAMMING T, APPENZELLER-HERZOG C. Destroy and exploit:catalyzed removal of hydroperoxides from the endoplasmic reticulum[J]. I J Cell Bio, 2013, 2013:180906.
[30] WOOD Z A, SCHRÖDER E, ROBIN HARRIS J, et al. Structure, mechanism and regulation of peroxiredoxins[J]. Trends Bio Sci, 2003, 28(1):32-40.
[31] SAARINEN M, GLEASON F K, EKLUND H. Crystal structure of thioredoxin-2 from Anabaena[J]. Structure, 1995, 3(10):1097-1108.
[32] MASUTANI H, YODOI J. Thioredoxin. Overview[J]. Method in Enzymol, 2002, 347:279-286.
[33] POWIS G, MONTFORT W R. Properties and biological activities of thioredoxins[J]. Annu Rev Biophys Biomol Struct, 2001, 30:421-455.
[34] PUIG A, LYLES M M, NOIVA R, et al. The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase[J]. J Bio Chem, 1994, 269(29):19128-19135.
[35] LAWRENCE R A, BURK R F. Glutathione peroxidase activity in selenium-deficient rat liver. 1976[J]. Biochem Biophys Res Commun, 2012, 425:503-509.
[36] 洪雅真,林晓凤,李继秋. 扇形游仆虫谷胱甘肽过氧化物酶基因克隆和序列特征分析[J].华南师范大学学报(自然科学版),2014,46(01):77-82.
HONG Y Z, LIN X F, LI J Q. Cloning and characterization of glutathione peroxidase gene from euplotes vannus[J]. Journal of South China Normal University (Natural Science Edition), 2014, 46(1):71-76.(in Chinese)
[37] 乔新荣. 植物谷胱甘肽过氧化物酶(GPX)研究进展[J].生物技术通报,2016,32(09):7-13.
[LL] QIAO X R. Research progress on GPX in plants[J]. Biotechnology Bulletin, 2016, 32(9):7-13.(in Chinese)
[38] DEAR T N, CAMPBELL K, RABBITTS T H. Molecular cloning of putative odorant-binding and odorant-metabolizing proteins[J]. Biochemistry, 1991, 30:10376-10382.
[39] SZTAJER H, GAMAIN B, AUMANN K D, et al. The putative glutathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase[J]. J Biol Chem, 2001, 276:7397-403.
[40] GAMAIN B, LANGSLEY G, FOURMAUX M N, et al. Molecular characterization of the glutathione peroxidase gene of the human malaria parasite Plasmodium falciparum[J]. Mol Biochem Parasitol, 1996, 78:237-248.
[41] COOKSON E, BLAXTER M L, SELKIRK M E. Identification of the major soluble cuticular glycoprotein of lymphatic filarial nematode parasites (gp29) as a secretory homolog of glutathione peroxidase[J]. Proc Natl Acad Sci U S A, 1992, 89:5837-5841.