蓝氏贾第鞭毛虫单克隆抗体的制备及鉴定

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (2): 160-163.

蓝氏贾第鞭毛虫单克隆抗体的制备及鉴定

章乐生^1,², 孙磊¹, 胡媛¹, 巩文词¹, 王燕娟¹, 沈玉娟¹, 徐馀信¹, 汪天平², 曹建平^1,^*()

1 中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,上海 200025
2 安徽省血吸虫病防治研究所,合肥 230061

收稿日期:2017-01-26 出版日期:2017-04-20 发布日期:2017-05-02
通讯作者: 曹建平
基金资助:
国家公益性卫生行业科研专项（No. 201502021);国家自然科学基金（No. 81371841）;上海市公共卫生第四轮三年行动计划（No. 15GWZK0101,GWIV-29)

Preparation and identification of monoclonal antibodies targeting Giardia lamblia

Le-sheng ZHANG^1,², Lei SUN¹, Yuan HU¹, Wen-ci GONG¹, Yan-juan WANG¹, yu-juan SHEN¹, Yu-xin XU¹, Tian-ping WANG², Jian-ping CAO^1,^*()

1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Key Laboratory of Parasite and Vector Biology, Ministry of Health; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai 200025, China
2 Anhui Institute of Schistosomiasis Control, Hefei 230061, China

Received:2017-01-26 Online:2017-04-20 Published:2017-05-02
Contact: Jian-ping CAO
Supported by:
Supported by the National Special Program for Scientific Research of Public Health of China (No. 201502021), the National Natural Science Foundation of China(No. 81371841) and the Fourth Round of Three-Year Public Health Action Plan of Shanghai, China (No. 15GWZK0101 and GWIV-29)

摘要/Abstract

摘要：

目的制备并鉴定蓝氏贾第鞭毛虫（简称贾第虫,Giardia lamblia）单克隆抗体（mAb）。方法以贾第虫滋养体抗原免疫2只8周龄雌性BALB/c小鼠,利用细胞融合技术建立抗贾第虫抗原杂交瘤细胞株并制备mAb。制备贾第虫滋养体可溶性抗原（STA）和排泄-分泌抗原（ESP）,采用免疫球蛋白及亚型检测试剂盒鉴定mAb的类型及亚型。蛋白质印迹(Western blotting)鉴定mAb对贾第虫滋养体STA和ESP的识别。采用ELISA检测mAb与贾第虫滋养体抗原反应性,并检测与其他寄生虫抗原的交叉反应。结果获得12株分泌贾第虫单克隆抗体细胞株（BB3、CE5、CC10、EG4、GC7、CC1、EF6、DB8、BG10、GH7、HC7和EB2）,均为IgG1亚型。其中EB2能识别贾第虫滋养体STA和ESP中相对分子质量分别为175 000和191 000的蛋白条带,与大肠埃希菌（Escherichia coli）、猪蛔虫（Ascaris suum）、人芽囊原虫（Blastocystis hominis）、日本血吸虫（Schistosoma japonicum）虫卵、日本血吸虫成虫和卫氏并殖吸虫（Paragonimus westermani）成虫抗原等均无交叉反应。结论制备的贾第虫特异性mAb为IgG1亚型。

关键词: 蓝氏贾第鞭毛虫, 滋养体, 单克隆抗体, 酶联免疫吸附试验, 蛋白质印迹

Abstract:

Objective To prepare and identify the monoclonal antibodies (mAb) against Giardia lamblia.Methods Two BALB/c mice of 8 weeks were immunized with antigens extracted from G. lamblia trophozoites, based on which mAb was prepared with traditional hybridoma technology. mAb isotyping was performed using enzyme-linked immunosorbent assay (ELISA) with coatings by soluble trophozoites antigen (STA) and excretory-secretory protein (ESP) prepared from G. lamblia trophozoites. mAb recognition of STA and ESP was identified by Western blotting. ELISA was performed to detect mAb reactions with G. lamblia trophozoite antigen as well as its cross-reactions with antigens from other parasite species. Results Twelve mAb-secreting hybridoma cell lines were obtained, comprising BB3, CE5, CC10, EG4, GC7, CC1, EF6, DB8, BG10, GH7, HC7 and EB2, all producing the IgG1-subtype mAb. The EB2 mAb specifically recognized proteins with M_r of 175 000 and 191 000 from STA and ESP, respectively, with no cross-reactions to the antigen extracts from Escherichia coli, Ascaris suum, Blastocystis hominis, eggs and adult worms of Schistosoma japonicum, and adult worms of Paragonimus westermani. Conclusion The specific IgG1-type mAb for G. lamblia trophozoites has been prepared.

Key words: Giardia lamblia, Trophozoite, Monoclonal antibody, ELISA, Western blotting

中图分类号:

R382.213

章乐生, 孙磊, 胡媛, 巩文词, 王燕娟, 沈玉娟, 徐馀信, 汪天平, 曹建平. 蓝氏贾第鞭毛虫单克隆抗体的制备及鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 160-163.

Le-sheng ZHANG, Lei SUN, Yuan HU, Wen-ci GONG, Yan-juan WANG, yu-juan SHEN, Yu-xin XU, Tian-ping WANG, Jian-ping CAO. Preparation and identification of monoclonal antibodies targeting Giardia lamblia[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(2): 160-163.

图/表 3

参考文献 20

[1]	Gardner TB, Hill DR.Treatment of giardiasis[J]. Clin Microbiol Rev, 2001, 14(1): 114-128.
[2]	Brodsky RE, Spencer HC Jr, Schultz MG.Giardiasis in American travelers to the Soviet Union[J]. J Infect Dis, 1974, 130(3): 319-323.
[3]	余源, 李巍伟, 陈阳, 等. 蓝氏贾第鞭毛虫细胞骨架蛋白的分子生物学研究进展[J]. 中国病原生物学杂志, 2013, 8(12): 1140-1141, 1147.
[4]	Quintero J, Figueroa DC, Barcelo R, et al. Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice[J]. Mem Inst Oswaldo Cruz, 2013, 108(5): 616-622.
[5]	宋百军, 刘原源, 宫鹏涛, 等. 双抗夹心ELISA检测犬粪贾第虫抗原方法的建立及应用[J]. 中国病原生物学杂志, 2014, 9(10): 902-904.
[6]	Feng Y, Xiao L.Zoonotic potential and molecular epidemiology of Giardia species and giardiasis[J]. Clin Microbiol Rev, 2011, 24(1): 110-140.
[7]	Keister DB.Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile[J]. Trans R Soc Trop Med Hyg, 1983, 77(4): 487-488.
[8]	Gottstein B, Harriman GR, Conrad JT, et al. Antigenic variation in Giardia lamblia: cellular and humoral immune response in a mouse model[J]. Parasite Immunol, 1990, 12(6): 659-673.
[9]	Jiménez JC, Morelle W, Michalsky JC, et al. Excreted/secreted glycoproteins of G. intestinalis play an essential role in the antibody response[J]. Parasitol Res, 2007, 100(4): 715-720.
[10]	Liu A, Yang F, Shen Y, et al. Genetic analysis of the Gdh and Bg genes of animal-derived Giardia duodenalis isolates in Northeastern China and evaluation of zoonotic transmission potential[J]. PLoS One, 2014, 9(4): e95291.
[11]	Zhang W, Shen Y, Wang R, et al. Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission[J]. PLoS One, 2012, 7(2): e31262.
[12]	Zhang W, Zhang X, Wang R, et al. Genetic characterizations of Giardia duodenalis in sheep and goats in Heilongjiang Province, China and possibility of zoonotic transmission[J]. PLoS Negl Trop Dis, 2012, 6(9): e1826.
[13]	Liu H, Shen Y, Yin J, et al. Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China[J]. BMC Infect Dis, 2014, 14: 25.
[14]	刘华, 沈玉娟, 张玉梅, 等. 临床腹泻患者贾第虫感染和分子特征研究[J]. 中国血吸虫病防治杂志, 2015, 27(2): 152-155.
[15]	袁忠英, 姜岩岩, 何祖安, 等. 湖北赤壁市农村儿童感染贾第虫的初步分析[J]. 国际医学寄生虫病杂志, 2015, 42(2): 86-88.
[16]	许隆祺, 蒋则孝. 我国人体寄生虫的虫种概况[J]. 中国寄生虫学与寄生虫病杂志, 1997, 15(5): 311-313.
[17]	Sandhu H, Mahajan RC, Ganguly NK.Flowcytometric assessment of the effect of drugs on Giardia lamblia trophozoites in vitro[J]. Mol Cell Biochem, 2004, 265(1-2): 151-160.
[18]	卢潍媛, 袁忠英, 沈玉娟, 等. 环介导等温扩增技术检测蓝氏贾第鞭毛虫[J]. 国际医学寄生虫病杂志, 2010, 37(3): 145-147.
[19]	顾有方, 汪凯, 刘德义, 等. 宠物犬蓝氏贾第鞭毛虫和隐孢子虫感染的分子检测[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(5): 362-367.
[20]	俞英昉, 吴秀萍, 储言红, 等. 安徽农村地区HIV感染者和幼儿园儿童蓝氏贾第鞭毛虫感染情况及其基因型[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(6): 537-541.