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

目的筛选并制备亚洲带绦虫45 kDa雌激素调节蛋白(Ta EP45)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Ta EP45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Ta EP45的反应原性。以纯化的Ta EP45免疫BALB/c小鼠(100μg/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Ta EP45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Ta EP45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(Mr)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Ta EP45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Ta EP45,筛选出5株能稳定分泌高滴度Ta EP45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。

Objective To clone and express 45 kDa Taenia asiatica estrogen-regulated protein(Ta EP45) as a diagnostic antigen for detecting infection of T. asiatica. Methods The open reading frame of Ta EP45 was amplified from the total c DNA of T. asiatica using specific primers designed from Ta EP45 sequence deposited in GenBank,then subcloned into the prokaryotic expression vector pET30 a(+). The recombinant plasmid DNA was transformed into Escherichia coli BL21(DE3) cells for expression. The recombinant Ta EP45 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 Ta EP45 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 Ta EP45. The recombinant Ta EP45 was highly expressed in E. coli BL21 cells with relative molecular mass of 60 000. The purified recombinant Ta EP45 was strongly recognized by serum from pig infected with T. asiatica. After being immunized with recombinant Ta EP45 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 Ta EP45 with titers higher than 1 ∶ 2 430. The subtype antibodies were IgG2 b or IgG1. All McAbs were able to recognize T. asiatica worm lysates and recombinant Ta EP45 as well.Conclusion The Ta EP45 was successfully cloned from T. asiatica cDNA and the recombinant Ta EP45 protein was expressed in E. coli. Five hybridoma cell lines that stably secreted McAbs against Ta EP45 are obtained with high titer of antibodies, which are able to recognize T. asiatica worm lysates.