刚地弓形虫磷酸丙糖异构酶多克隆抗体的制备及鉴定分析

doi:10.12140/j.issn.1000-7423.2019.03.012

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (3): 311-316.doi: 10.12140/j.issn.1000-7423.2019.03.012

刚地弓形虫磷酸丙糖异构酶多克隆抗体的制备及鉴定分析

赵正虎¹(), 钱胜南², 万静宜³, 唐子茹³, 沈双^1,^*()

1 上海健康医学院附属第六人民医院东院,上海 201306
2 上海海洋大学,上海 201306
3 上海健康医学院,上海 201306

收稿日期:2019-03-12 出版日期:2019-06-30 发布日期:2019-07-10
通讯作者: 沈双
作者简介:
作者简介：赵正虎（1986-）,男,本科,检验技师,从事临床检验。E-mail: 14390778@qq.com
基金资助:
上海健康医学院教学建设项目（No. 2018037）;上海健康医学院种子基金（No. SFP-18-22-14-009）;浦东新区科技发展基金（No. PKJ2018-Y55）;上海市卫生和计划生育委员会青年项目（No. 20174Y0124）

Preparation of polyclonal antibody against-triosephosphate isomerase of Toxoplasma gondii and its application to detect T. gondii infection

Zheng-hu ZHAO¹(), Sheng-nan QIAN², Jing-yi WAN³, Zi-ru TANG³, Shuang SHEN^1,^*()

1 Shanghai Sixth People’s Hospital East Affiliated to Shanghai University of Medicine and Health Sciences, Shanghai 201306, China;
2 Shanghai Ocean University, Shanghai 201306, China
3 Shanghai University of Medicine and Health Sciences, Shanghai 201306, China

Received:2019-03-12 Online:2019-06-30 Published:2019-07-10
Contact: Shuang SHEN
Supported by:
Supported by Teaching Construction Projects of Shanghai University of Medicine and Health Sciences (No. 2018037); the Seed Fund Program of Shanghai University of Medicine and Health Sciences (No. SFP-18-22-14-009); Pudong New Area Science and Technology Development Fund (No. PKJ2018-Y55) and Youth Project of Shanghai Municipal Commission of Health and Family Planning (No. 20174Y0124)

摘要/Abstract

摘要：

目的研制刚地弓形虫磷酸丙糖异构酶（TPI）兔多克隆抗体（多抗）并分析其诊断价值。方法在大肠埃希菌BL21重组表达菌表达TPI,镍柱亲和层析法纯化后与弗氏佐剂等体积混合,颈背部多点注射免疫新西兰大白兔（TPI 200 μg/次,共3次）。末次免疫后2周收集兔血清,Protein A亲和层析纯化多抗。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析多抗纯度,ELISA分析多抗效价,蛋白质印迹（Western blotting）及ELISA分析多抗识别弓形虫排泄分泌抗原及纯化TPI的效果。利用多抗偶联磁珠,富集11份弓形虫感染小鼠和11份健康小鼠血清后,Dot-blot分析多抗的诊断价值,并与弓形虫循环抗原ELISA试剂盒的检测结果作比较。结果表达并纯化获得条带单一的TPI蛋白。SDS-PAGE分析结果显示,免疫兔血清经Protein A亲和纯化后获得的多抗,重链与轻链清晰可见。第3次免疫后兔血清抗体效价可达1 : 280 000以上;纯化后的多抗纯度高,效价可达1 : 100 000以上;Western blotting分析结果显示,纯化的多抗能识别弓形虫排泄分泌抗原中的TPI及纯化的TPI,在相对分子质量（M_r）28 000处均出现一特异性条带;ELISA分析结果显示,纯化的多抗能结合纯化的TPI,其A₄₅₀值与TPI蛋白的包被浓度呈正相关。Dot-blot结果显示,11份感染鼠血清中10份鉴定为阳性,1份阴性;11份健康小鼠血清中10份鉴定为阴性,1份假阳性。弓形虫循环抗原ELISA试剂盒检测结果显示,11份感染小鼠血清中6份鉴定为阳性,5份阴性;11份健康小鼠血清均鉴定为阴性。Dot-blot与ELISA试剂盒检测结果差异无统计学意义（P > 0.05）。结论制备并纯化了弓形虫TPI多抗,该多抗具有潜在诊断价值,可用于开发免疫诊断试剂以检测弓形虫感染。

关键词: 刚地弓形虫, 磷酸丙糖异构酶, 多克隆抗体, 诊断

Abstract:

Objective Triosephosphate isomerase (TPI) of Toxoplasma gondii is an immunodominant antigen secreted by T. gondii during infection and therefore an important diagnostic antigen. To explore its potential for diagnostic purpose, the polyclonal antibody anti-TPI was made in rabbit and its application to detect T. gondii infection was explored. Methods A New Zealand white rabbit was immunized with 200 μg recombinant TPI protein expressed in Escherichia coli and emulsified with Freund’s adjuvant for three times. The serum was collected from the immunized rabbit 2 weeks after the last immunization. The total IgG in immunized-rabbit serum was purified with Protein A affinity purification column. SDS-PAGE was used to detect the purity of the purified IgG. The anti-TPI antibody titers in the purified IgG and in the immune sera was measured by ELISA and its specific recognition of recombinant TPI and native TPI in T. gondii excretory/secretory (ES) products was detected by Western blotting. The anti-TPI IgG was conjugated on magnet beats to pull down the TPI in T. gondii-infected mouse sera and the pull-downed TPI was applied on NC membrane to be detected by the anti-TPI IgG, and the results were compared with that detected by ELISA kit. Results The recombinant TPI protein was expressed in E. coli and the purified TPI protein was used to immunize a rabbit. The rabbit anti-TPI serum was obtained and the total IgG in the serum was purified with Protein-A affinity column. SDS-PAGE analysis identified that the purified IgG contained typical IgG heavy chain and light chain. The anti-TPI specific antibody titers were 1 : 128 000 in immunized rabbit serum and in the purified IgG measured by ELISA. Western blotting showed that the recombinant TPI protein and native TPI in T. gondii ES products could be specifically recognized by the purified IgG. The purified rabbit IgG was able to pull down the TPI antigen in 10 of 11 sera of mice infected with T. gondii, however, one of the 11 normal mouse sera also showed positive. The results detected by ELISA kit showed that 6 of the 11 infected mice were positive and 5 were negative; 11 healthy mice were all negative. There was no significant difference in the results between Dot blot and ELISA detection. Conclusion The anti-TPI polyclonal antibody was prepared in immunized rabbit. An anti-TPI IgG-based immunological assay was potentially established to detect T. gondii infection. This TPI-specific IgG could be used to develop an immunodiagnostic assay to detect T. gondii infection.

Key words: Toxoplasma gondii, Triosephosphate isomerase, Polyclonal antibody, Diagnosis

中图分类号:

R382.5

赵正虎, 钱胜南, 万静宜, 唐子茹, 沈双. 刚地弓形虫磷酸丙糖异构酶多克隆抗体的制备及鉴定分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(3): 311-316.

Zheng-hu ZHAO, Sheng-nan QIAN, Jing-yi WAN, Zi-ru TANG, Shuang SHEN. Preparation of polyclonal antibody against-triosephosphate isomerase of Toxoplasma gondii and its application to detect T. gondii infection[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(3): 311-316.

图/表 6

图1

图2

图3

图4

图5

图6

参考文献 21

[1]	Dubey JP, Jones JL.Toxoplasma gondii infection in humans and animals in the United States[J]. Int J Parasitol, 2008, 38(11): 1257-1278.
[2]	Pereira KS, Franco RM, Leal DA.Transmission of toxoplasmosis (Toxoplasma gondii) by foods[J]. Adv Food Nutr Res, 2010, 60: 1-19.
[3]	Dubey JP.The history of Toxoplasma gondii: the first 100 years[J]. J Eukaryot Microbiol, 2008, 55(6): 467-475.
[4]	马远林, 吕芳丽. Ⅰ型干扰素在原虫感染中的作用[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 399-404.
[5]	Nissapatorn V.Toxoplasmosis in HIV/AIDS: a living legacy[J]. Southeast Asian J Trop Med Public Health, 2009, 40(6): 1158-1178.
[6]	Gao XJ, Zhao ZJ, He ZH, et al. Toxoplasma gondii infection in pregnant women in China[J]. Parasitology, 2012, 139(2): 139-147.
[7]	Wang H, Wang T, Luo QL, et al. Prevalence and genotypes of Toxoplasma gondii in pork from retail meat stores in Eastern China[J]. Int J Food Microbiol, 2012, 157(3): 393-397.
[8]	Garcia JL, Navarro IT, Vidotto O, et al. Toxoplasma gondii: comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs[J]. Exp Parasitol, 2006, 113(2): 100-105.
[9]	Rostami A, Karanis P, Fallahi S.Advances in serological, imaging techniques and molecular diagnosis of Toxoplasma gondii infection[J]. Infection, 2018, 46(3): 303-315.
[10]	Nam HW.GRA proteins of Toxoplasma gondii: maintenance of host-parasite interactions across the parasitophorous vacuolar membrane[J]. Korean J Parasitol, 2009, 47(Suppl): S29-S37.
[11]	Shwab EK, Jiang TT, Pena HF, et al. The ROP18 and ROP5 gene allele types are highly predictive of virulence in mice across globally distributed strains of Toxoplasma gondii[J]. Int J Parasitol, 2016, 46(2): 141-146.
[12]	沈双, 肖迪, 宋丽君, 等. 应用免疫蛋白质组学方法鉴定弓形虫病诊断抗原分子[J]. 中国病原生物学杂志, 2015, 10(2): 152-158, 164.
[13]	沈双, 殷旭仁, 宋丽君, 等. 弓形虫磷酸丙糖异构酶原核表达及免疫保护的初步研究[J]. 中国血吸虫病防治杂志, 2017, 29(6): 780-783.
[14]	Bringaud F, Rivière L, Coustou V.Energy metabolism of trypanosomatids: adaptation to available carbon sources[J]. Mol Biochem Parasitol, 2006, 149(1): 1-9.
[15]	Abrahamsen MS, Templeton TJ, Enomoto S, et al. Complete genome sequence of the apicomplexan, Cryptosporidium parvum[J]. Science, 2004, 304(5669): 441-445.
[16]	Bhowmick IP, Kumar N, Sharma S, et al. Plasmodium falciparum enolase: stage-specific expression and sub-cellular localization[J]. Malar J, 2009, 8: 179.
[17]	Ginger ML, McFadden GI, Michels PAM. Rewiring and regulation of cross-compartmentalized metabolism in protists[J]. Phil Trans R Soc B, 2010, 365(1541): 831-845.
[18]	Rider SD Jr, Zhu G.Cryptosporidium: genomic and biochemical features[J]. Exp Parasitol, 2010, 124(1): 2-9.
[19]	孙铭飞, 吴彩艳, 戚南山, 等. 顶复门原虫糖酵解代谢及其关键酶: 己糖激酶的研究进展[J]. 畜牧与兽医, 2011, 43(11): 89-92.
[20]	Kotresha D, Noordin R.Recombinant proteins in the diagnosis of toxoplasmosis[J]. APMIS, 2010, 118(8): 529-542.
[21]	Liesenfeld O, Montoya JG, Kinney S, et al. Effect of testing for IgG avidity in the diagnosis of Toxoplasma gondii infection in pregnant women: experience in a US reference laboratory[J]. J Infect Dis, 2001, 183(8): 1248-1253.

刚地弓形虫磷酸丙糖异构酶多克隆抗体的制备及鉴定分析

Preparation of polyclonal antibody against-triosephosphate isomerase of Toxoplasma gondii and its application to detect T. gondii infection

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

[1]	景天宇, 牟汝涛, 张帆, 刘现兵, 胡雪梅, 张海霞, 李志丹. 孕期刚地弓形虫感染对滋养层细胞和蜕膜免疫细胞表面CD73表达的影响及其与不良妊娠的关系[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 210-216.
[2]	章孝成, 胡媛, 彭荟, 沈玉娟, 刘华, 曹建平. 基于CRISPR/Cas9技术的刚地弓形虫bfd2缺陷虫株的构建及表型分析[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 217-222.
[3]	张铃, 刘星卓, 吕芳丽. 非洲刚地弓形虫感染和弓形虫病流行概况[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 241-251.
[4]	贾西帅, 周水茂, 罗华堂, 刘聪, 杨燕, 田俊华, 徐文秀. 湖北省1例输入性内脏利什曼病的诊断与分析[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 298-300.
[5]	殷荷, 马磊, 党甜甜, 李佳铭, 赵志军. 刚地弓形虫Ⅰ/Ⅲ型ROP16调控TAF15对THP-1细胞影响及机制研究[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(1): 103-111.
[6]	郭嘉天, 林琳, 卢艳. 曼氏裂头蚴病诊断技术研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(6): 776-782.
[7]	李仕毓, 李静, 陆绍红, 郑斌. 宿主细胞自主免疫抗弓形虫的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(5): 653-658.
[8]	谢俏, 黎俊, 董丽凤. 10例阿米巴肠炎患者的临床特征和内镜下特征[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(4): 496-501.
[9]	马莹莹, 彭金华, 吴宁, 张冬民, 郑惠平. 2017–2023年河南省许昌市输入性疟疾特征及病例诊断分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(4): 529-532.
[10]	何伊莎, 谢朝勇, 王毓, 李燕菁. 南京市新型冠状病毒感染疫情暴发前后输入性疟疾流行特征及病例诊断分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(2): 267-271.
[11]	解晓曼, 孙航, 代莉莎, 朱文菊, 王利磊, 谢环环, 董宏杰, 张俊梅, 王琦, 周贝贝, 赵桂华, 徐超, 尹昆. 刚地弓形虫感染对小鼠脑组织转录本m⁶A甲基化修饰的影响[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 27-35.
[12]	郑广福, 刘现兵, 姜昱竹, 李新雨, 胡雪梅, 张海霞. 刚地弓形虫感染孕鼠胎盘组织中中性粒细胞和IL-17与不良妊娠结局的关系[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 48-54.
[13]	薛羽珊, 林萍, 程训佳, 冯萌. 慢性弓形虫感染对宿主中枢神经系统的损伤及其作用机制[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(5): 527-531.
[14]	姜文静, 孟雅莉, 赵利娜, 王春苗, 张晓磊. 刚地弓形虫棒状体蛋白18和膜表面抗原30复合核酸疫苗对小鼠的免疫保护作用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(5): 532-538.
[15]	王登辉, 张艳, 聂蔷, 刘红威. 结肠小袋纤毛虫感染1例[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(5): 647-649.