副流感病毒5型SH蛋白单克隆抗体的制备

Preparation of Monoclonal Antibody against SH Protein of Parainfluenza Virus 5

关键词:  副流感病毒5型  SH蛋白  单克隆抗体

Key words:  PIV5  SH protein  monoclonal antibody

摘要:

为获得副流感病毒5型SH蛋白的单克隆抗体，将构建的重组表达质粒pET43a-SH转化BL21(DE3)plys，经IPTG诱导表达、过镍柱纯化后将重组蛋白作为免疫原，免疫8周龄Balb/c雌性小鼠，并按常规方法制备杂交瘤细胞。通过间接免疫荧光方法进行筛选，获得3株杂交瘤细胞株，命名为5B9、3G8、4E11，并进行了培养特性、分泌抗体活性、分泌抗体亚类的鉴定。结果显示：3株细胞株连续传代10代均稳定分泌单克隆抗体，细胞上清液IFA效价稳定，分泌单克隆抗体亚型均为IgG2a。制备并纯化了1株(5B9)单克隆抗体，浓度为4.26 mg/mL，纯度不低于90%，IFA效价不低于1∶2000，与猪牛等常见病毒均无交叉反应。

Abstract:

In order to obtain the monoclonal antibody against SH protein of parainfluenza virus 5 (PIV5), the recombinant expression plasmid pET43a-SH was transformed into BL21 (DE3) plys. The recombinant plasmid pET43a-SH was induced by IPTG, purified by nickel column and used as immunogen to immunize 8-week-old female Balb/c mice, and hybridoma cells were prepared by conventional methods. Three hybridoma cell lines, named 5B9, 3G8 and 4E11, were obtained by indirect immunofluorescence method. Their culture characteristics, antibody secreting activity and subclass were identified. The results showed that the three cell lines stably secreted monoclonal antibodies for 10 passages, and the IFA titer of cell supernatant was stable, and the subtype of secreted monoclonal antibodies was IgG2a. A strain of (5B9) monoclonal antibody mouse ascites was prepared and purified,which concentration was 4.26 mg / mL, the purity was not less than 90%,the IFA titer was not less than 1∶2000，and the monoclonal antibodies showed no cross reaction with common animal viruses by IFA.