禽流感病毒NP蛋白单克隆抗体的制备与鉴定

Preparation of Monoclonal Antibody against NP Protein of H9N2 Avian influenza virus

关键词:  H9N2亚型禽流感病毒  NP蛋白  单克隆抗体

Key words:  H9N2 Avian influenza,  NP protein  monoclonal antibody

摘要:

为获得H9N2亚型禽流感病毒（Avian influenza virus，AIV）NP蛋白的单克隆抗体，将构建的重组表达质粒pET-30a-NP转化BL21细胞，经IPTG诱导表达、纯化后作为免疫原，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ELISA方法、Western-blot方法进行筛选，获得3株杂交瘤细胞株，命名为3A6、2H12、5F7，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，分泌单克隆抗体亚型均为IgG2b。制备并纯化了以上3株单克隆抗体，浓度分别为2.9、2.5、2.8mg/mL，纯度不低于90%，与禽常见病毒均无交叉反应。

Abstract:

In order to obtain the monoclonal antibody against NP protein of H9N2 Avian influenza, the recombinant expression plasmid pET30a-NP was transformed into BL21 cells. The recombinant plasmid pET30a-NP was induced by IPTG, purified 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 3A6, 2H12 and 5F7, were obtained by ELISA, Western-blot and IFA 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 subtype of secreted monoclonal antibodies was IgG2b. The three strains of monoclonal antibody mouse ascites was prepared and purified,which concentration were 2.9, 2.5, 2.8 mg / mL separately, the purity were not less than 90%, and the monoclonal antibodies showed no cross reaction with common avain animal viruses by Western-blot.