亚洲带绦虫45 kDa雌激素调节蛋白的表达及其杂交瘤细胞株的筛选与鉴定

doi:10.12140/j.issn.1000-7423.2019.01.013

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (1): 70-74.doi: 10.12140/j.issn.1000-7423.2019.01.013

亚洲带绦虫45 kDa雌激素调节蛋白的表达及其杂交瘤细胞株的筛选与鉴定

王立群(), 梁盼红, 张少华, 侯俊玲, 王莉杰, 毛立, 骆学农^*()

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

收稿日期:2018-08-30 出版日期:2019-02-28 发布日期:2019-03-18
通讯作者: 骆学农
作者简介:
作者简介：王立群（1994-）,女,硕士研究生,从事人兽共患病与兽医公共卫生学。E-mail: 1282690114@qq.com
基金资助:
国家重点研发计划（No. 2017FYD0501303）;国家自然科学基金（No. 31772726）

Cloning and expression of 45 kDa Taenia asiatica estrogen-regulated protein and development of monoclonal antibodies

Li-qun WANG(), Pan-hong LIANG, Shao-hua ZHANG, Jun-ling HOU, Li-jie WANG, Li MAO, Xue-nong LUO^*()

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

Received:2018-08-30 Online:2019-02-28 Published:2019-03-18
Contact: Xue-nong LUO
Supported by:
Supported by National Key Research and Development Program(No. 2017FYD0501303) and National Nature Science Foundation of China(No. 31772726)

摘要/Abstract

摘要：

目的筛选并制备亚洲带绦虫45 kDa雌激素调节蛋白（TaEP45）的杂交瘤细胞株。方法设计带有酶切位点的特异性引物,以亚洲带绦虫成虫总RNA为模板,RT-PCR扩增TaEP45完整的开放阅读框(ORF),克隆至原核表达载体pET-30a(+),转化至大肠埃希菌BL21(DE3),用1 mmol/L 异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达带组氨酸（His）标签的TaEP4蛋白,用亚洲带绦虫囊尾蚴阳性猪血清蛋白质印迹（Western blotting）分析TaEP45的反应原性。以纯化的TaEP45免疫BALB/c小鼠（100 μg/鼠,首次免疫用等量弗氏完全佐剂,第2、第3次免疫用等量弗氏不完全佐剂）,选择血清抗体效价高的小鼠,取脾细胞和SP2/0骨髓瘤细胞融合,用纯化的TaEP45和His标签蛋白ELISA筛选阳性杂交瘤细胞株,用亚洲带绦虫全虫抗原Western blotting鉴定杂交瘤细胞株分泌的单克隆抗体。结果 RT-PCR扩增获得的TaEP45基因ORF长1 362 bp,编码453个氨基酸。重组融合蛋白的相对分子质量（M_r）约为60 000,可被亚洲带绦虫囊尾蚴阳性猪血清识别。筛选获得5株稳定分泌TaEP45抗体的杂交瘤细胞株,其抗体效价均≥ 1 : 2 430,亚型分别为IgG2b或IgG1。Western blotting分析结果显示,5株杂交瘤细胞株分泌的单克隆抗体均能与亚洲带绦虫全虫抗原发生反应。结论表达纯化了TaEP45,筛选出5株能稳定分泌高滴度TaEP45单克隆抗体的杂交瘤细胞株,其分泌的单克隆抗体能与亚洲带绦虫全虫抗原发生反应。

关键词: 亚洲带绦虫, 45 kDa雌激素调节蛋白, 杂交瘤细胞株

Abstract:

Objective To clone and express 45 kDa Taenia asiatica estrogen-regulated protein (TaEP45) as a diagnostic antigen for detecting infection of T. asiatica. Methods The open reading frame of TaEP45 was amplified from the total cDNA of T. asiatica using specific primers designed from TaEP45 sequence deposited in GenBank, then subcloned into the prokaryotic expression vector pET30a(+). The recombinant plasmid DNA was transformed into Escherichia coli BL21（DE3） cells for expression. The recombinant TaEP45 with His-tag was expressed in E. coli under induction of 1 mmol/L isopropyl β-D-thiogalactoside(IPTG) and purified with nickel column. The purified recombinant TaEP45 was tested for its recognition by serum from pig infected with T. asiatica by Western blotting, and then used to immunize BALB/c mice to make monoclonal antibody (McAb). Results RT-PCR amplified a gene product of 1 362 bp which encodes 453 amino acids of TaEP45. The recombinant TaEP45 was highly expressed in E. coli BL21 cells with relative molecular mass of 60 000. The purified recombinant TaEP45 was strongly recognized by serum from pig infected with T. asiatica. After being immunized with recombinant TaEP45 formulated with Freud’s adjuvant, the splenocytes of mice were used to make hybridoma cell lines. Total 5 hybridoma cell lines were obtained that stably produced McAbs against TaEP45 with titers higher than 1 : 2 430. The subtype antibodies were IgG2b or IgG1. All McAbs were able to recognize T. asiatica worm lysates and recombinant TaEP45 as well. Conclusion The TaEP45 was successfully cloned from T. asiatica cDNA and the recombinant TaEP45 protein was expressed in E. coli. Five hybridoma cell lines that stably secreted McAbs against TaEP45 are obtained with high titer of antibodies, which are able to recognize T. asiatica worm lysates.

Key words: Taenia asiatica, 45 kDa estrogen-regulated protein, Hybridoma cell strains

中图分类号:

R383.32

王立群, 梁盼红, 张少华, 侯俊玲, 王莉杰, 毛立, 骆学农. 亚洲带绦虫45 kDa雌激素调节蛋白的表达及其杂交瘤细胞株的筛选与鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1): 70-74.

Li-qun WANG, Pan-hong LIANG, Shao-hua ZHANG, Jun-ling HOU, Li-jie WANG, Li MAO, Xue-nong LUO. Cloning and expression of 45 kDa Taenia asiatica estrogen-regulated protein and development of monoclonal antibodies[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(1): 70-74.

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杂交瘤细胞株 Cell lines	培养上清不同稀释度的A₄₅₀值 A₄₅₀ value of different dilutions of culture supernatants						阴性对照 Negative control	抗体效价 Antibody titer	抗体亚型 Isotype of McAbs	抗体浓度 /μg·ml^-1 Antibody concentration /μg·ml^-1
杂交瘤细胞株 Cell lines	1 : 10	1 : 30	1 : 90	1 : 270	1 : 810	1 : 2 430	阴性对照 Negative control	抗体效价 Antibody titer	抗体亚型 Isotype of McAbs	抗体浓度 /μg·ml^-1 Antibody concentration /μg·ml^-1
2C11A3	2.590	1.464	2.226	1.696	0.993	0.461	0.069	1 : 2 430	IgG2b,κ	8.405
5C11D3	2.468	2.359	2.142	1.715	1.052	0.511	0.069	1 : 2 430	IgG2b,κ	8.843
4B95H	2.214	2.029	1.628	1.210	0.732	0.367	0.069	1 : 2 430	IgG1,κ	6.058
8E7D5	2.599	2.527	2.204	1.533	0.801	0.352	0.069	1 : 2 430	IgG1,κ	6.948
10E6D4	2.571	2.560	2.422	2.014	1.370	0.705	0.069	1 : 2 430	IgG2b,κ	8.704

亚洲带绦虫45 kDa雌激素调节蛋白的表达及其杂交瘤细胞株的筛选与鉴定

Cloning and expression of 45 kDa Taenia asiatica estrogen-regulated protein and development of monoclonal antibodies

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