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

›› 2016, Vol. 43 ›› Issue (2): 304-310.doi: 10.16431/j.cnki.1671-7236.2016.02.003

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

赵洪喜^1,2, 刘军龙¹, 杨聪山¹, 赵帅阳¹, 刘娟¹, 刘光远¹, 殷宏^1,3, 关贵全¹, 罗建勋¹

1. 中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 甘肃省动物寄生虫病重点实验室, 兰州 730046;
2. 宁夏大学农学院, 银川 750021;
3. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2015-08-26 出版日期:2016-02-20 发布日期:2016-03-02
通讯作者: 关贵全, 罗建勋 E-mail:guanguiquan@163.com;ljxbn@163.com
作者简介:赵洪喜(1977-),男,内蒙古赤峰人,博士生,研究方向:动物寄生虫及其分子生物学,E-mail:zhaohongxi2006@163.com
基金资助:
国家重点基础研究发展计划(973计划)(2015CB150300);国家自然科学基金项目(31372432、31402189);农业科技创新工程(ASTIP);PiroVAC (KBBE-3-245145);948项目(2010-S06);国家肉牛牦牛产业技术体系(NBCIS CARS-38)

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

ZHAO Hong-xi^1,2, LIU Jun-long¹, YANG Cong-shan¹, ZHAO Shuai-yang¹, LIU Juan¹, LIU Guang-yuan¹, YIN Hong^1,3, GUAN Gui-quan¹, LUO Jian-xun¹

1. Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. College of Agriculture, Ningxia University, Yinchuan 750021, China;
3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease, Yangzhou 225009, China

Received:2015-08-26 Online:2016-02-20 Published:2016-03-02

摘要/Abstract

摘要： 为了研究环形泰勒虫3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的功能,本试验利用PCR技术从环形泰勒虫裂殖体cDNA中扩增环形泰勒虫GAPDH (TaGAPDH)基因,将扩增产物克隆到原核表达载体pET-30a(+)中,并转入大肠杆菌BL21(DE3)中进行融合蛋白诱导表达,融合蛋白纯化后免疫家兔,制备多克隆抗体,分别用间接ELISA和Western blotting检测抗体的效价和特异性;利用激光共聚焦荧光显微镜观察TaGAPDH蛋白在环形泰勒虫裂殖体中的亚细胞定位.结果显示,克隆得到了TaGAPDH全长基因,大小为1 020 bp;SDS-PAGE分析结果显示,融合蛋白大小约44 ku,且以包涵体形式存在.制备的多克隆抗体效价高达1:12 800,具有很高的特异性;亚细胞定位显示TaGAPDH蛋白主要分布于环形泰勒虫裂殖体细胞质内.以上结果表明本试验成功克隆表达了TaGAPDH基因,并制备了针对TaGAPDH蛋白的兔多克隆抗体,为筛选环形泰勒虫病疫苗、药物靶点及研究环形泰勒虫能量代谢奠定了基础.

关键词: 环形泰勒虫; 3-磷酸甘油醛脱氢酶; 多克隆抗体

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

中图分类号:

赵洪喜, 刘军龙, 杨聪山, 赵帅阳, 刘娟, 刘光远, 殷宏, 关贵全, 罗建勋. 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[J]. , 2016, 43(2): 304-310.

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.

参考文献

[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.

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

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.
[3]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[4]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[5]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[6]	牛小杰, 徐明国, 肖莉, 刘文豪, 陈创夫, 王勇. 鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(3): 1218-1228.
[7]	张梦杨, 何健, 刘阳坤, 姚伦广. 非洲猪瘟病毒p37蛋白生物信息学分析及其多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(1): 300-309.
[8]	朱丽霖, 王后坤, 陈雪阳, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡IFIT5对J亚群禽白血病病毒在DF-1细胞内复制的影响[J]. 中国畜牧兽医, 2023, 50(1): 310-316.
[9]	肖洋洋, 马忠臣, 李芮芮, 陈创夫, 郑炜, 王勇, 王鹏雁. 布鲁氏菌分泌蛋白BspI生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(8): 3112-3121.
[10]	王炜, 韩慧慧, 丁乃峥, 何成强. 牛病毒性腹泻病毒E0蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(8): 3131-3139.
[11]	刘铭, 张永宁. 猪丁型冠状病毒N蛋白原核表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2022, 49(7): 2698-2707.
[12]	许春梅, 邵明珠, 王心悦, 林佳迪, 郭建雄, 张祥胤, 童德文, 梁瑞英, 赵晓民. 非洲猪瘟病毒D250R蛋白生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(12): 4745-4755.
[13]	刘超, 王后坤, 朱丽霖, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡RNF165蛋白多克隆抗体制备及其生物信息学分析[J]. 中国畜牧兽医, 2022, 49(11): 4159-4167.
[14]	张天爱, 李婷婷, 陶晓莉, 李藤菲, 林家锋, 胡思漫, 刘文秀, 佟伟, 李永刚. 猪流行性腹泻病毒S蛋白原核表达及多克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2022, 49(11): 4383-4391.
[15]	杨景, 张新宇, 梁志鹏, 程晴, 王聪颖, 池仕红, 袁生, 郭锦玥, 黄淑坚, 温峰. H9N2亚型禽流感病毒NP蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(10): 3963-3971.