环形泰勒虫3-磷酸甘油醛脱氢酶的原核表达、多克隆抗体制备及亚细胞定位

doi:10.16431/j.cnki.1671-7236.2016.02.003

Abstract

Abstract: In order to study the function of the Theileria annulata (T.annulata) glyceraldehyde-3-phosphate dehydrogenase (GAPDH),the T.annulata GAPDH (TaGAPDH) gene was amplified by PCR from the cDNA of T.annulata,and cloned into the pET-30a(+) vector and expressed in Escherichia coli BL21 (DE3).After purification,the fusion protein was injected into rabbits to produce polyclonal antibodies.Specificity and titers of the polyclonal antibodies were determined by ELISA and Western blotting,and subcellular localization of TaGAPDH protein was observed by confocal fluorescence microscope.The results showed that TaGAPDH gene was approximately 1 020 bp;SDS-PAGE analysis showed that the fusion protein was expressed in BL21(DE3) with 44 ku in molecular mass,and highest expressed level was detected in the inclusion.The titer of the polyclonal antibodies was more than 1:12 800.The results of western blotting indicated that the polyclonal antibodies possessed good specificity,and TaGAPDH protein was predominantly present on the cytoplasm of T.annulata schizont by subcellular localization.This study laid the foundation for screening and identifying candidate antigens of vaccination and drug targets as well as studying the energy metabolism of T.annulata.

Key words: Theileria annulata; glyceraldehyde-3-phosphate dehydrogenase; polyclonal antibody

CLC Number:

ZHAO Hong-xi, LIU Jun-long, YANG Cong-shan, ZHAO Shuai-yang, LIU Juan, LIU Guang-yuan, YIN Hong, GUAN Gui-quan, LUO Jian-xun. Prokaryotic Expression,Polyclonal Antibody Preparation and Subcellular Localization of Theileria annulata GAPDH[J]. , 2016, 43(2): 304-310.

References

[1] 孔繁瑶.家禽寄生虫学[M].北京:中国农业大学出版社,1997.
[2] Chaussepied M,Janski N,Baumgartner M,et al.TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility[J].PLoS Pathogens,2010,6(11):e1001197.
[3] 郑进峰,罗金,谢俊仁,等.环形泰勒虫裂殖体表面蛋白基因PCR诊断方法的建立[J].中国畜牧兽医,2014,41(1):47-51.
[4] 吕文祥,吕文顺,张其才,等.我国牛蜱传性血液原虫的虫种调查和分布特征的研究[J].中国兽医科技,1995,25(8):13-16.
[5] Seitzer U,Ahmed J.Tropical theileriosis:Cytotoxic T lymphocyte response to vaccination[J].Vaccine,2008,26 (Suppl 6):G24-G28.
[6] 曹雯丽,王真,沙它尔·卡哈尔,等.牛环形泰勒虫裂殖子/梨浆虫表面抗原基因非疏水区的克隆及GST-P27重组蛋白的高效表达[J].中国畜牧兽医,2014,41(3):49-53.
[7] Morrison W I,Mckeever D J.Current status of vaccine development against Theileria parasites[J].Parasitology,2006,133(Suppl):S169-S187.
[8] 张然然,刘华淼,邢秀梅,等.鹿茸组织中内参基因的筛选和验证[J].中国畜牧兽医,2015,42(4):883-889.
[9] 尚海旭,井然,贾弘禔,等.GAPDH功能多样性[J].生理科学进展,2011,30(5):371-374.
[10] 岑双庆,李全有,李亚琼,等.环形泰勒虫微线体－棒状体蛋白的亚细胞定位分析[J].中国兽医科学,2014,44(8):800-804.
[11] 廖申权,戚南山,吴彩艳,等.顶复门原虫3-磷酸甘油醛脱氢酶功能及其应用研究进展[J].中国畜牧兽医,2012,39(1):71-74.
[12] Allsopp B A,Gibson W C,Stagg D A.Characterization of some East African Theileria species isolates by isoenzyme analysis,with particular reference to T.parva[J].International Journal for Parasitology,1985,15(3):271-276.
[13] van Dooren G G,Stimmler L M,Mcfadden G I.Metabolic maps and functions of the Plasmodium mitochondrion[J].Fems Microbiology Ecology,2006,30(4):596-630.
[14] Rickard M D.Carbohydrate metabolism in Babesia rodhaini:Differences in the metabolism of normal and infected rat erythrocytes[J].Experimental Parasitology,1969,25(1):16-31.
[15] Pfaff N,Gross U,Bohne W.Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii[J].International Journal for Parasitology,2008,38(10):1121-1132.
[16] Verlinde C L,Callens M,Van calenbergl S,et al.Selective inhibition of trypanosomal glyceraldehyde-3-phosphate dehydrogenase by protein structure-based design:Toward new drugs for the treatment of sleeping sickness[J].Medicinal Chemistry,1994,37(21):3605-3613.
[17] Senkovich O,Speed H,Grigorian A,et al.Crystallization of three key glycolytic enzymes of the opportunistic pathogen Cryptosporidium parvum[J].Biochim Biophys Acta,2005,1750(2):166-172.
[18] Sundararaj K P,Wood R E,Ponnusamy S,et al.Rapid shortening of telomere length in response to ceramide involoves the inhibition of telomere binding activity of nuclear glyceraldehyde-3-phosphate dehydrogenase[J].Journal of Biological Chemistry,2004,9(7):152-6162.
[19] Zhou Y,Yi X,Stoffer J B,et al.The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer[J].Molecular Cancer Research,2008,6(8):1375-1384.
[20] Kondo S,Kubota S,Mukudai Y,et al.Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 Mrna[J].Biochemlcal and Biophysical Research Communications,2011,405(3):382-387.
[21] Sen N,Hara M R,Kornberg M D,et al.Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis[J].Nature Cell Biology,2008,10(7):866-873.
[22] Argirl L,Henri S,Dessein H,et al.Induction of a protective immunity against Schistosoma mansoni with ovalbumin-coupled Sm37-5 coadsorbed with granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-2 on alum[J].Vaccine,1999,17(1):13-18.
[23] 闫玉涛,李小红,刘述先,等.日本血吸虫三磷酸甘油醛脱氢酶核酸疫苗小鼠体内的免疫应答特征[J].中国寄生虫学与寄生虫病杂志,2005,23(5):266-269.
[24] Waine G J,Becker M,Yang W,et al.Cloning,molecular characterization,and function activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase,a putative vaccine candidate against Schistosomiasis japonica[J].Infection and Immunity,1993,61(11):4716-4723.
[25] Hannaert V,Blaauw M,Kohl L,et al.Molecular analysis of the cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase in Leishmania mexicana[J].Molecular and Biochemical Parasitology,1992,55(1-2):115-126.
[26] Akinyi S,Gaona J,Meyer E V,et al.Phylogenetic and structural information on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in Plasmodium provides functional insights[J].Infection Genetics and Evolution.2008,8(2):205-212.
[27] Singh S,Malik B K,Sharma D K.Molecular modeling and docking analysis of Entamoeba histolytica glyceraldehyde-3 phosphate dehydrogenase,a potential target enzyme for anti-protozoal drug development[J].Chem Biol Drug Des,2008,71(6):554-562.
[28] Micheis P A,Marchand M,Kohl L,et al.The cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase in Trypanosoma brucei have a distant evolutionary relationship[J].Eur J Biochem,1991,198(2):421-428.

Prokaryotic Expression,Polyclonal Antibody Preparation and Subcellular Localization of Theileria annulata GAPDH

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[3]	WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.
[4]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[5]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[6]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[7]	ZHANG Mengyang, HE Jian, LIU Yangkun, YAO Lunguang. Bioinformatics Analysis and Polyclonal Antibody Preparation of African Swine Fever Virus p37 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 300-309.
[8]	ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.
[9]	XIAO Yangyang, MA Zhongchen, LI Ruirui, CHEN Chuangfu, ZHENG Wei, WANG Yong, WANG Pengyan. Bioinformatic Analysis and Polyclonal Antibody Preparation of Brucella Secreted Protein BspI [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3112-3121.
[10]	LIU Ming, ZHANG Yongning. Prokaryotic Expression of Porcine Deltacoronavirus Nucleocapsid Protein and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2698-2707.
[11]	XU Chunmei, SHAO Mingzhu, WANG Xinyue, LIN Jiadi, GUO Jianxiong, ZHANG Xiangyin, TONG Dewen, LIANG Ruiying, ZHAO Xiaomin. Bioinformatics Analysis of African Swine Fever Virus D250R Protein and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4745-4755.
[12]	LIU Chao, WANG Houkun, ZHU Lilin, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Polyclonal Antibody Preparation and Bioinformatics Analysis of Chicken RNF165 [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4159-4167.
[13]	ZHANG Tianai, LI Tingting, TAO Xiaoli, LI Tengfei, LIN Jiafeng, HU Siman, LIU Wenxiu, TONG Wei, LI Yonggang. Prokaryotic Expression of Porcine Epidemic Diarrhea Virus S Protein and Preparation and Identification of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4383-4391.
[14]	YANG Jing, ZHANG Xinyu, LIANG Zhipeng, CHENG Qing, WANG Congying, CHI Shihong, YUAN Sheng, GUO Jinyue, HUANG Shujian, WEN Feng. Prokaryotic Expression of NP Protein of H9N2 Subtype Avian Influenza Virus and Preparation of Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3963-3971.
[15]	ZHENG Huizhen, PU Hao, MIAO Ronghao, GAN Lu, GE Xiaomin, Bayinchahan, LI Yongchang, GUO Qingyong. Prokaryotic Expression and Bioinformatics Analysis of Theileria annulata enolase Gene [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3972-3981.