新城疫病毒M蛋白单克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2021.09.034

摘要/Abstract

摘要： 为获得新城疫病毒（Newcastle disease virus，NDV） M蛋白单克隆抗体，本研究将编码该蛋白的基因克隆、表达并纯化重组蛋白，作为免疫原免疫小鼠，同时建立ELISA筛选方法对小鼠血清效价进行测定，筛选出血清抗体效价最高的小鼠脾细胞与SP2/0细胞融合，最终获得能稳定产生NDV M蛋白单克隆抗体的杂交瘤细胞株，并进行了抗体的免疫荧光、Western blotting、亚类的鉴定、杂交瘤细胞染色体计数、小鼠腹水制备及腹水内单克隆抗体效价测定。结果显示，PCR、重组质粒测序及双酶切鉴定正确，M基因大小约为1 095 bp。SDS-PAGE和Western blotting检测显示，试验成功表达了重组M蛋白，分子质量约为60 ku，且可与NDV阳性血清反应。建立的ELISA筛选方法中，重组M蛋白、His标签蛋白和抗体的最佳工作浓度分别为0.5 μg/mL、0.5 μg/mL和1∶256 000。抗体的免疫荧光、Western blotting、亚类的鉴定显示，杂交瘤细胞产生的抗体可与NDV SG10株及重组M蛋白特异性结合，其轻链为κ，重链为IgG2A。C9-G2、D3-F2杂交瘤细胞染色体计数结果分别为97和101条；杂交瘤细胞上清的ELISA检测效价均为1∶6 400，腹水的ELISA检测效价分别为1∶409 600和1∶102 400。本研究成功制备了NDV M蛋白的单克隆抗体，可为进一步研究M蛋白的功能提供工具。

关键词: 新城疫病毒(NDV); M蛋白; 单克隆抗体

Abstract: To obtain the monoclonal antibodies against Newcastle disease virus (NDV) M protein, the gene encoding the protein was cloned, the recombinant protein was expressed and purified. Mice were immunized with recombinant M protein, and an appropriate ELISA screening method was established. After the serum titer of mice was determined by ELISA, the mouse spleen cells with the highest serum antibody titer were screened and fused with SP2/0 cells. Hybridoma cell lines that could stably produce monoclonal antibodies against NDV M protein were obtained. Immunofluorescence, Western blotting, subclass identification, chromosome count of hybridoma cells, preparation of mouse ascites, and titer determination of monoclonal antibody in ascites were carried out. The results of PCR, recombinant plasmid sequencing and double enzyme digestion showed that this study successfully amplified the NDV M gene, which was about 1 095 bp in size. SDS-PAGE and Western blotting analysis showed that the recombinant M protein was successfully expressed with about 60 ku molecular weight, and it could react with positive serum of NDV. In the established ELISA screening method, the optimal working concentration or dilution of recombinant M protein, His tagged protein and antibody were 0.5 μg/mL, 0.5 μg/mL and 1∶256 000, respectively. Immunofluorescence, Western blotting and subclass identification of the antibody showed that the hybridoma cells produced antibody could specifically bind to NDV SG10 strain and recombinant M protein, and its light chain was κ and heavy chain was IgG2A. Chromosome counts of C9-G2 and D3-F2 hybridioma cells were 97 and 101, respectively. The ELISA titers of the supernatant of the hybridioma cell lines were both 1∶6 400. And the ELISA titers of ascites were 1∶409 600 and 1∶102 400, respectively. In this study, monoclonal antibodies against NDV M protein were successfully prepared, which could provide tools for further study of the function of M protein.

Key words: Newcastle disease virus (NDV); M protein; monoclonal antibody

中图分类号:

S852.65⁺7

曾建宇, 董振远, 贾文凤, 张国中, 薛佳. 新城疫病毒M蛋白单克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2021, 48(9): 3423-3431.

ZENG Jianyu, DONG Zhenyuan, JIA Wenfeng, ZHANG Guozhong, XUE Jia. Preparation and Identification of Monoclonal Antibody Against Newcastle Disease Virus M Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3423-3431.

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