弓形虫胚层发育相关蛋白的原核表达及免疫原性研究

中国畜牧兽医

弓形虫胚层发育相关蛋白的原核表达及免疫原性研究

田维鹏¹^，2，张念章^{2，高琦²^，3，鲁力^{2，朱兴全²，宋铭忻¹}}

（1.东北农业大学动物医学学院，黑龙江哈尔滨 150030；2.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，甘肃兰州 730046；3.华南农业大学兽医学院，广东广州 510642）

收稿日期:2013-10-14 出版日期:2014-04-20 发布日期:2014-05-27
通讯作者: 宋铭忻（1968—），男，黑龙江人，博士生导师，从事兽医寄生虫学与寄生虫病学研究。E-mail：songmx@neau.edu.cn
作者简介:田维鹏（1988—），男，山东人，硕士生，研究方向：兽医寄生虫病学及分子生物学。
基金资助:
国家自然科学基金（31230073、31172316）；甘肃省创新研究群体计划项目（1210RJIA006）。

Study on Prokaryotic Expression and Immunogenicity of Toxoplasma gondii Embryogenesis-related Protein

TIAN Wei-peng¹, 2, ZHANG Nian-zhang², GAO Qi²^{, 3}, LU Li², ZHU Xing-quan², SONG Ming-xin¹

（1. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China; 2. Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Lanzhou 730046, China; 3. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China）

Received:2013-10-14 Online:2014-04-20 Published:2014-05-27

摘要/Abstract

摘要： 试验旨在研究弓形虫胚层发育相关蛋白（TgERP）的免疫原性，以弓形虫RH株的基因组DNA为模板，扩增TgERP基因，将其连接于表达载体pET-30a(+)后，转化至Escherichia coli BL21（DE3）感受态细胞进行IPTG诱导表达，应用Western blotting对重组蛋白的反应原性进行分析，并用纯化后的TgERP蛋白免疫新西兰大白兔，制备多克隆抗体。结果显示，在37 ℃条件下用1.0 mmol/L IPTG诱导6 h表达可溶性TgERP蛋白的量最大。SDS-PAGE结果显示，目的蛋白分子质量为16.7 ku，以可溶形式表达，纯化后蛋白条带单一。经Western blotting分析，TgERP蛋白有较好的反应原性；制备的多克隆抗体效价较高，可达1∶51200，表明该蛋白具有较好免疫原性。提示，TgERP蛋白可作为血清学诊断方法的候选抗原和弓形虫病疫苗的候选分子，为建立弓形虫新型诊断方法和研制新型弓形虫疫苗奠定了基础。

关键词:

弓形虫; 弓形虫胚层发育相关蛋白基因; 原核表达; 免疫原性

Abstract: In order to analyze the immunogenicity of Toxoplasma gondii embryogenesis-related protein （TgERP）, the TgERP gene was amplified from genomic DNA of T.gondii RH strain by PCR and then cloned into prokaryotic expression plasmid pET-30a (+). The recombinant plasmid pET-30-ERP was transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The expressed product was purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blotting. The polyclonal antibody against the recombinant protein was prepared by immunizing New Zealand White rabbits with the purified protein. PCR amplification and restriction analysis proved that the restriction prokaryotic expression plasmid pET-30a (+) was constructed correctly and sequencing results showed that the cloned TgERP gene was identified to be the corresponding sequence reported on the website （http: //toxodb.org）. The TgERP protein was expressed under 1.0 mmol/L IPTG at 37 ℃, shaking for 6 h. SDS-PAGE analysis showed the protein product was successfully purified with a molecular weight of about 16.7 ku. Western blotting and ELISA analyses indicated that the TgERP protein reacted with sera from vaccinated rabbit. In conclusion, it could be a potential candidate antigen for developing new detection methods or new sub-unit vaccine against toxoplasmosis.

Key words: Toxoplasma gondii; Toxoplasma gondii embryogenesis-related protein gene; prokaryotic expression; immunogenicity

田维鹏，张念章，高琦，鲁力，朱兴全，宋铭忻. 弓形虫胚层发育相关蛋白的原核表达及免疫原性研究[J]. 中国畜牧兽医.

TIAN Wei-peng, ZHANG Nian-zhang, GAO Qi, LU Li, ZHU Xing-quan, SONG Ming-xin. Study on Prokaryotic Expression and Immunogenicity of Toxoplasma gondii Embryogenesis-related Protein[J]. .

参考文献

1 Du F, Zhang Q, Yu Q, et al. Soil contamination of Toxoplasma gondii oocysts in pig farms in central China［J］. Vet Parasitol, 2012, 187: 53～56.
2 Dubey J P, Lunney J K, Shen S K, et al. Infectivity of low numbers of Toxoplasma gondii oocysts to pigs［J］. J Parasitol, 1996, 82: 438～443.
3 Dubey J P, Jones J L. Toxoplasma gondii infection in humans and animals in the United States［J］. Int J Parasitol, 2008, 38: 1257～1278.
4 Dubey J P. Toxoplasmosis of animals and humans［M］. Second Edition. Boca Raton: CRC Press, Florida, 2010.
5 Fayer R, Dubey J P, Lindsay D S. Zoonotic protozoa: From land to sea［J］. Trends Parasitol, 2004, 20: 531～536.
6 Fritz H M, Bowyer P W, Bogyo M, et al. Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance［J］. PLoS One, 2012, 1: 1～14.
7 Hill D, Coss C, Dubey J P, et al. Identification of a sporozoite-specific antigen from Toxoplasma gondii ［J］. J Parasitol, 2011, 97: 328～337.
8 Hundertmark M, Hincha D K. LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana ［J］. BMC Genomics, 2008, 9: 118.
9 Jones J L, Dubey J P. Foodborne toxoplasmosis［J］. Clin Infect Dis, 2012, 55: 845～851.
10 Lass A, Pietkiewicz H, Modzelewska E, et al. Detection of Toxoplasma gondii oocysts in environmental soil samples using molecular methods［J］. Eur J Clin Microbiol Infect Dis, 2009, 28: 599～605.
11 Lélu M, Villena I, Dardé M L, et al. Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil［J］. Appl Environ Microbiol, 2012, 78: 5127～5132.
12 Rajapakse S, Chrishan S M, Samaranayake N, et al. Antibiotics for human toxoplasmosis: A systematic review of randomized trials［J］. Pathog Glob Health, 2013, 107: 162～169.
13 Simon A, Poulin M B, Rousseau A N, et al. Fate and transport of Toxoplasma gondii oocysts in seasonally snow covered watersheds: A conceptual framework from a melting snowpack to the Canadian arctic coasts［J］. Int J Environ Res Public Health, 2013, 10: 994～1005.
14 Torrey E F, Yolken R H. Toxoplasma oocysts as a public health problem［J］. Trends Parasitol, 2013, 29: 380～384.
15 Van Wormer E, Fritz H, Shapiro K, et al. Molecules to modeling: Toxoplasma gondii oocysts at the human-animal-environment interface［J］. Comp Immunol Microbiol Infect Dis, 2013, 36: 217～231.
16 Wainwright K E, Miller M A, Barr B C, et al. Chemical inactivation of Toxoplasma gondii oocysts in water［J］. J Parasitol, 2007a, 93: 925～931.
17 Wainwright K E, Lagunas-Solar M, Miller M A, et al. Physical inactivation of Toxoplasma gondii oocysts in water［J］. Appl Environ Microbiol, 2007b, 73: 5663～5666.
18 Zhou P, Chen Z, Li H L, et al. Toxoplasma gondii infection in humans in China［J］. Parasit Vectors, 2011, 4: 165.

[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]	李灿, 李楚楚, 曾维, 张宁, 纪春晓, 陈韬. 猪RegⅢγ蛋白的重组表达及功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2414-2426.
[3]	宋越, 苏胜杰, 张帆, 白帆, 戴伶俐, 王娜, 王大伟, 杨茜雯, 赵世华, 张月梅. 溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2479-2486.
[4]	张芳源, 蔺雅婷, 杨大为, 陈虎, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立[J]. 中国畜牧兽医, 2023, 50(5): 2012-2022.
[5]	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.
[6]	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.
[7]	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.
[8]	刘家兴, 韩雪英, 张鑫茹, 邓嘉昕, 姚伦广, 刘阳坤. 携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析[J]. 中国畜牧兽医, 2023, 50(4): 1567-1574.
[9]	贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 细粒棘球绦虫EGR-07243和EGR-07244基因生物信息学分析及原核表达[J]. 中国畜牧兽医, 2023, 50(3): 847-858.
[10]	章志涛, 李祥龙, 孔令丽, 罗雨昕, 王冬英. 大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A的原核表达及不同时期表达水平分析[J]. 中国畜牧兽医, 2023, 50(3): 1107-1117.
[11]	甘露, 郑会珍, 诺明达来, 刘燕, 金敏, 何文文, 温丽翠, 李永畅, 巴音查汗·盖力克. 伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析[J]. 中国畜牧兽医, 2023, 50(2): 469-478.
[12]	吕双飞, 马勋, 王静, 孔翠莲, 寇丽君, 刘彩霞, 史唯地, 康立超, 任慧杰. 炭疽芽孢杆菌PA-LF1融合蛋白的原核表达及其反应原性研究[J]. 中国畜牧兽医, 2023, 50(2): 674-683.
[13]	张凯, 张春平, 戈胜强, 孙春喜, 程杰, 伏春雨, 王刚, 牛星, 彭军. 非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(1): 270-279.
[14]	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.
[15]	王宇琛, 田广原, 周雅坪, 郭婷, 赵红梅, 孙雅婕, 赵逢淼, 卞宇晨, 于佳梁, 郝永清. BPIV3 NP蛋白的原核表达及其对BPIV3灭活疫苗免疫增强作用的研究[J]. 中国畜牧兽医, 2022, 49(8): 3122-3130.