中华蜜蜂MRJP7基因的原核表达及多克隆抗体的制备

Prokaryotic Expression of mrjp7 of Apis cerana cerana and Preparation of Its Polyclonal Antibody

用PCR方法从含中华蜜蜂Accmrjp7基因的质粒DNA模板中扩增出MRJP7成熟肽编码区片段,将该片段克隆入原核表达载体pGEX-4T-2,在大肠杆菌(Escherichia coli)BL21(DE3)中进行融合表达,SDS-PAGE和薄层扫描显示,该基因得到高效表达,表达产物大小约为66 kDa,占细胞总蛋白的22.8%。利用割胶回收的方法,获得目的蛋白,注射兔子制备多克隆抗体,并利用所获抗体进行Western blot印迹分析,检测MRJPs,获得特异性条带。

The coding region of MRJP7 mature peptide was amplified by PCR from Apis cerana cerana eDNA library and cloned into the prokaryotic expression vector pGEX-4T-2 for fusion expression in E. coli. The SDS-PAGE and thin-layer scanning results showed that the expressed GST-MRJP7 fusion protein was about 66 kDa in size and about 22.8% in proportion to the total bacterial proteins. The expressed products were retrieved from the SDS-PAGE gels and used to immunize the rabbit to produce the polyclonal antibody against MRJPs. The titer of the antibody was evaluated by ELISA method and its specificity was confirmed by Western blot analysis.