宿主GAPDH对柔嫩艾美耳球虫子孢子入侵细胞的影响

doi:10.11843/j.issn.0366-6964.2019.10.015

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (10): 2088-2096.doi: 10.11843/j.issn.0366-6964.2019.10.015

宿主GAPDH对柔嫩艾美耳球虫子孢子入侵细胞的影响

王璐¹, 朱顺海¹, 赵其平¹, 黄兵¹, 韩红玉¹, 刘桂玲^1,2, 李志行^1,2, 赵焕之¹, 董辉^1*

1. 中国农业科学院上海兽医研究所农业部动物寄生虫学重点实验室, 上海 200241;
2. 上海师范大学生命与环境科学学院, 上海 200234

收稿日期:2019-05-05 出版日期:2019-10-23 发布日期:2019-10-23
通讯作者: 董辉,主要从事兽医寄生虫分子生物学与寄生虫病免疫学等研究,E-mail:donghui@shvri.ac.cn
作者简介:王璐(1993-),女,河南三门峡人,硕士生,主要从事兽医寄生虫分子生物学研究
基金资助:
国家重点研发计划（2017YFD0500400）；国家寄生虫种质资源共享服务平台（平台-TDRC-22）

Effects of Host Glyceraldehyde-3-phosphate Dehydrogenase on Eimeria tenella Sporozoites Invasion of Cells

WANG Lu¹, ZHU Shunhai¹, ZHAO Qiping¹, HUANG Bing¹, HAN Hongyu¹, LIU Guiling^1,2, LI Zhihang^1,2, ZHAO Huanzhi¹, DONG Hui^1*

1. Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
2. College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China

Received:2019-05-05 Online:2019-10-23 Published:2019-10-23

摘要/Abstract

摘要： 目前，关于宿主细胞应对球虫感染机制的研究极其有限。在前期研究中，我们利用相对和绝对定量同位素标记技术结合液相色谱串联质谱技术分析了感染柔嫩艾美耳球虫子孢子72 h后鸡胚成纤维（CEF）细胞的蛋白质组表达变化，作者拟进一步研究其中一个上调蛋白——甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase，GAPDH）对柔嫩艾美耳球虫子孢子入侵细胞的影响。克隆鸡GAPDH基因并构建原核重组质粒pGEX-4T-1-GAPDH，表达重组GAPDH蛋白，用Western blot对表达进行验证。使用q-PCR、Western blot和免疫组织化学的方法检测感染柔嫩艾美耳球虫子孢子后DF-1细胞中GAPDH的表达量，并使用抗体抑制试验检测GAPDH多克隆抗体对柔嫩艾美耳球虫子孢子入侵细胞的影响。结果显示：成功克隆1 123 bp的GAPDH基因，并获得61.7 ku的重组GAPDH蛋白。q-PCR和免疫组织化学检测均显示，GAPDH在柔嫩艾美耳球虫子孢子感染的DF-1细胞中的表达显著升高。抗体抑制试验表明，与相同浓度的正常兔IgG相比，50、100、200、300和400 μg·mL^-1的抗GAPDH抗体均能明显抑制子孢子的入侵。以上结果表明，子孢子感染会引起宿主细胞GAPDH蛋白的上调，并且宿主GAPDH蛋白在子孢子入侵过程中起正调控作用。

Abstract: At present, the mechanism of how the host cell responds to Eimeria tenella infection is highly limited. In our previous study, isobaric tags for relative and absolute quantitation were used to study the proteomics changes in host cells after infection with E. tenella sporozoites. In this study, one of the up-regulated proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was selected to study its effect on the invasion of Eimeria tenella sporozoites. Gallus gallus GAPDH gene was cloned, and the prokaryotic recombinant expression plasmid pGEX-4T-1-GAPDH was constructed. The recombinant GAPDH protein (rGAPDH) was expressed and identified by Western blot. Expression of GAPDH in DF-1 cells during E. tenella sporozoites infection was detected by q-PCR, Western blot and immunohistochemistry. The effect of GAPDH polyclonal antibody on sporozoites invasion of cells was detected by antibody inhibition assay. Results were as follows:A 1 123-bp GAPDH was cloned and the rGAPDH was recognized as a~61.7 kDa protein by Western blot. The expression of GAPDH in E. tenella sporozoites-infected DF-1 cells were up-regulated by q-PCR and immunohistochemistry detection. The antibody inhibition assay showed that antibodies against GAPDH at 50, 100, 200, 300 and 400 μg·mL^-1 could significantly inhibit sporozoite invasion compared with the same does of normal rabbit IgG. These results show that E. tenella sporozoites infection could induce the up-regulation of host GAPDH, and host GAPDH plays an active role in Eimeria invasion.

中图分类号:

S852.723

王璐, 朱顺海, 赵其平, 黄兵, 韩红玉, 刘桂玲, 李志行, 赵焕之, 董辉. 宿主GAPDH对柔嫩艾美耳球虫子孢子入侵细胞的影响[J]. 畜牧兽医学报, 2019, 50(10): 2088-2096.

WANG Lu, ZHU Shunhai, ZHAO Qiping, HUANG Bing, HAN Hongyu, LIU Guiling, LI Zhihang, ZHAO Huanzhi, DONG Hui. Effects of Host Glyceraldehyde-3-phosphate Dehydrogenase on Eimeria tenella Sporozoites Invasion of Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2088-2096.

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参考文献 23

[1]	BLAKE D P, TOMLEY F M. Securing poultry production from the ever-present Eimeria challenge[J]. Trends Parasitol, 2014, 30(1):12-19.
[2]	WILLIAMS R B. A compartmentalised model for the estimation of the cost of coccidiosis to the world's chicken production industry[J]. Int J Parasitol, 1999, 29(8):1209-1229.
[3]	周新锐, 田志英,古少鹏,等. 鸡球虫内生发育研究概况[J]. 畜牧兽医科技信息, 2009(10):4-5.
	ZHOU X R, TIAN Z Y, GU S P, et al. A survey of endophytic development of chicken coccidia[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2009(10):4-5. (in Chinese)
[4]	SAM-YELLOWE T Y. Rhoptry organelles of the apicomplexa:their role in host cell invasion and intracellular survival[J]. Parasitol Today, 1996, 12(8):308-316.
[5]	RAMLY N Z, ROUZHEINIKOV S N, SEDELNIKOVA S E, et al. Crystallization and preliminary crystallographic analysis of a surface antigen glycoprotein, SAG19, from Eimeria tenella[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2013, 69(12):1380-1383.
[6]	JIANG L L, LIN J J, HAN H Y, et al. Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella[J]. Parasitol Res, 2012, 110(2):865-874.
[7]	YAN M, CUI X X, ZHAO Q P, et al. Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella[J]. Parasite, 2018, 25:60.
[8]	ZHANG H L, GUO F G, ZHU G. Involvement of host cell integrin α2 in Cryptosporidium parvum infection[J]. Infect Immun, 2012, 80(5):1753-1758.
[9]	SCHMID M, HEITLINGER E, SPORK S, et al. Eimeria falciformis infection of the mouse caecum identifies opposing roles of IFNγ-regulated host pathways for the parasite development[J]. Mucosal Immunol, 2014, 7(4):969-982.
[10]	王璐,朱顺海,赵其平,等. 基于iTRAQ技术分析柔嫩艾美耳球虫感染后CEF细胞的差异表达蛋白[J/OL]. 中国动物传染病学报, 1-11. (2019-03-14). http://kns.cnki.net/kcms/detail/31.2031.s.20190313.1710.010.html. WANG L, ZHU S H, ZHAO Q P, et al. Analysis of differentially expressed proteins in CEF cells infected with Eimeria tenella by iTRAQ technology[J/OL]. Chinese Journal of Animal Infectious Diseases, 1-11. (2019-03-14). http://kns.cnki.net/kcms/detail/31.2031.s.20190313.1710.010.html. (in Chinese)
[11]	ZHANG J Y, ZHANG F, HONG C Q, et al. Critical protein GAPDH and its regulatory mechanisms in cancer cells[J]. Cancer Biol Med, 2015, 12(1):10-22.
[12]	COLELL A, GREEN D R, RICCI J E. Novel roles for GAPDH in cell death and carcinogenesis[J]. Cell Death Differ, 2009, 16(12):1573-1581.
[13]	SHEOKAND N, MALHOTRA H, KUMAR S, et al. Moonlighting cell-surface GAPDH recruits apotransferrin to effect iron egress from mammalian cells[J]. J Cell Sci, 2014, 127(19):4279-4291.
[14]	WAN G Q, YANG K, LIM Q, et al. Identification and validation of reference genes for expression studies in a rat model of neuropathic pain[J]. Biochem Biophys Res Commun, 2010, 400(4):575-580.
[15]	GUO C M, LIU S Q, SUN M Z. Novel insight into the role of GAPDH playing in tumor[J]. Clin Transl Oncol, 2013, 15(3):167-172.
[16]	GRILLON E, FARION R, REUVENI M, et al. Spatial profiles of markers of glycolysis, mitochondria, and proton pumps in a rat glioma suggest coordinated programming for proliferation[J]. BMC Res Notes, 2015, 8:207.
[17]	HAO L H, ZHOU X, LIU S Q, et al. Elevated GAPDH expression is associated with the proliferation and invasion of lung and esophageal squamous cell carcinomas[J]. Proteomics, 2015, 15(17):3087-3100.
[18]	GANNON J, OHLENDIECK K. Subproteomic analysis of basic proteins in aged skeletal muscle following offgel pre-fractionation[J]. Mol Med Rep, 2012, 5(4):993-1000.
[19]	付国良, 黄晓红. 甘油醛-3-磷酸脱氢酶功能的研究进展[J]. 生物物理学报, 2013, 29(3):181-191.
	FU G L, HUANG X H. Progress on research of functions of glyceraldehyde-3-phosphate dehydrogenase[J]. Acta Biophysica Sinica, 2013, 29(3):181-191. (in Chinese)
[20]	安欢迎, 王琴,李治,等. 家蚕微孢子虫与家蚕ECH1和GAPDH蛋白的相互作用[J]. 昆虫学报, 2017, 60(1):45-52.
	AN H Y, WANG Q, LI Z, et al. Interaction between Nosema bombycis and proteins BmECH1 and BmGAPDH in Bombyx mori[J]. Acta Entomologica Sinica, 2017, 60(1):45-52. (in Chinese)
[21]	LAO Y M, LU Y, JIANG J G, et al. Six regulatory elements lying in the promoter region imply the functional diversity of chloroplast GAPDH in Duanliella bardawil[J]. J Agric Food Chem, 2012, 60(36):9211-9220.
[22]	GAO J P, WANG D, CAO L Y, et al. Transcriptome sequencing of Codonopsis pilosula and identification of candidate genes involved in polysaccharide biosynthesis[J]. PLoS One, 2015, 10(2):e0117342.
[23]	索勋,李国清. 鸡球虫病学[M]. 北京:中国农业大学出版社, 1998.
	SUO X, LI G Q. Coccidia and coccidiosis of domestic fowl[M]. Beijing:China Agricultural University Press, 1998. (in Chinese)

宿主GAPDH对柔嫩艾美耳球虫子孢子入侵细胞的影响

Effects of Host Glyceraldehyde-3-phosphate Dehydrogenase on Eimeria tenella Sporozoites Invasion of Cells

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