施马伦贝格病毒核衣壳蛋白单克隆抗体的制备及其特性分析

Preparation and characterization of a monoclonal antibody against the nucleocapsid protein of Schmallenberg virus

目的制备施马伦贝格病毒(SBV)核衣壳蛋白(N)的单克隆抗体(mAb)并分析其特性。方法将SBV N基因分别构建至pET-28a-c(+)和pMAL-c5X载体中,在大肠杆菌中诱导表达带组氨酸标签的融合蛋白His-SBV-N和带麦芽糖结合蛋白(MBP)标签的融合蛋白MBP-SBV-N,分别用镍-次氮基三乙酸(Ni-NTA)树脂和直链淀粉树脂纯化两种蛋白;用纯化的His-SBVN免疫BALB/c小鼠制备mAb,以MBP-SBV-N为包被抗原间接ELISA筛选分泌mAb的杂交瘤细胞和测定mAb效价,利用蛋白G琼脂糖凝胶从腹水中纯化mAb;应用Western blot和间接免疫荧光分析mAb的反应性和特异性。结果筛选并纯化出1株抗SBV的mAb(命名为1F2),效价为1∶32 000,重链亚型为IgG2α,轻链为κ;1F2既能与重组SBV N蛋白发生反应,又能识别SBV,但与布尼亚病毒科内亲缘关系较近的沙门达病毒、道格拉斯病毒和赤羽病病毒的N蛋白存在交叉反应,而与裂谷热病毒N蛋白无交叉反应。结论成功制备了抗SBV核衣壳蛋白的mAb,为SBV的血清学检测提供了工具。

Objective The present study was conducted to prepare and characterize a monodonal antibody （rnAb） against the nucleocapsid （N） protein of Schmallenberg virus （SBV）. Methods The SBV N gene was cloned into pET-28a-c（＋） and pMAL-cSX vectors and then transformed into E. coli BL21. HisUdine （His）-tagged （His-SBV-N） and maltose-binding protein （MBP）-tagged （MBP-SBV-N） fusion proteins were respectively induced to express by IPTG and purified by nickel- nitrilotriacetic acid （Ni-NTA） agarose and amylose resin. His-SBV-N was used to immunize BALB/c mice to prepare mAb, and MBP-SBV-N was used as the coating antigen in ELISA to screen mAb-secreting hybridomas and to determine mAb titers. The mAb against SBV N protein was purified from the ascitic fluids using protein G sepharose. We, stem blotting and indirect immunofluorescence assay were utilized to analyze the reactivity and specificity of the mAb. Results One mAb specific for SBV N protein （named 1F2） was successfully screened and purified. The titer of 1F2 was 1：32 000. Besides, the isotype of 1F2 was determined to be IgG2α/K. 1F2 reacted with both recombinant SBV N proteins and SBV isolates. It was also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein. Conclusion One mAb specific for the SBV N protein was successfully prepared, it provides a useful tool for the serological detection of SBV.