牛IFN-γ原核表达、单克隆抗体制备及其ELISA检测方法的建立

Preparation of Monoclonal Antibodies Against Recombinant Bovine IFN-gamma and Development of Sandwich ELISA for Bovine IFN-gamma Detection

摘要:

实验旨在建立牛重组IFN-γ（BovIFN-γ）的ELISA检测技术，为牛传染病的免疫学诊断提供新方法。PHA刺激体外培养的奶牛外周血白细胞，从培养细胞中提取总RNA, 经过RT-PCR扩增出BovIFN-γ基因cDNA，进一步克隆至pET28a，转化大肠杆菌，经IPTG诱导，表达出预期大小（18kD左右）组氨酸标记蛋白，经鉴定为BovIFN-γ；以纯化的重组BovIFN-γ为免疫原，应用淋巴细胞杂交瘤技术，获得4株能稳定分泌抗BovIFN-γ单克隆抗体的细胞株，分别命名为A7、 A10、G6与G10。免疫球蛋白亚类鉴定证明杂交瘤细胞所分泌的抗体均为IgG₁，腹水效价在1∶2¹⁰×100～1∶2¹¹×100之间。Western-blot分析显示，4株单抗均能特异性结合重组BovIFN-γ。ELISA试验表明，4株单抗只与融合蛋白BovIFN-γ反应，而不与非相关性蛋白Ag85B、ESAT-6-CFP-10、GM-CSF等发生反应。选取A10细胞株分泌的单克隆抗体、纯化的多克隆抗体及辣根过氧化物酶（HRP）标记的羊抗兔IgG，建立了检测BovIFN-γ的双抗体夹心ELISA方法。实验结果表明，此方法检测敏感性达到2ng/mL，特异性良好，为进一步建立灵敏、特异的病原感染诊断方法奠定了基础。

Abstract:

This study was aimed to establish ELISA for recombinant bovine IFN-γ (BovIFN-γ) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-γ (BovIFN-γ) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-γ. The pETBovIFN-γ was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-γ as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-γ were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG₁. Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-γ, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-γ specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-γ, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-γ paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.