锯缘青蟹呼肠孤病毒p40蛋白的体外表达与单克隆抗体制备

锯缘青蟹呼肠孤病毒(Scylla serrata reovirus,SsRV)第9节段(S9)编码的p40蛋白可能为病毒的甲基转移酶(methyltransferase)。为了便于确认p40在病毒复制过程中扮演的角色,将S9片段的开放阅读框(ORF)克隆到原核表达载体pET28a(+),实现了在宿主表达菌E.coli BL21(DE3)中的高效表达,进而纯化了重组表达蛋白p40(rp40),并制备了抗SsRV p40蛋白的2株单克隆抗体(4B7、3E5)。经Western blot分析表明,2株单克隆抗体均可特异地识别rp40蛋白,以及识别SsRV病毒蛋白中分子量为46 ku和40 ku 2条蛋白条带。实验结果不但确认SsRV p40为SsRV的结构蛋白,也提示p40和p46蛋白可能具有相同的抗原表位。为该蛋白的后续功能研究奠定了基础。

In vitro expression of Scylla serrata reovirus protein p40 and preparation of monoclonal antibodies against the expressed protein

We previously suggested that the protein p40 ecoded by Scylla serrata reovirus (SsRV) segments S9 is likely to represent a Methyltransferase. Towards to simplifying to study the role of p40 in the SsRV replication, the open reading frame (ORF) of SsRV segments S9 was cloned into the pET 28a( ) expression vectors and introduced into E.coli BL21(DE3) by transformation. After induction, N-terminally 6×his-tagged p40 recobinant protein (rp40) with molecular masses of 46 kDa were obtained. The rp40 protein was purified by Ni-NTA and used for immunization of BALB/c mice for monoclonal antibody (MAb) production. Western blot assay showed that two MAbs could specifically recognize the rp40 expressed in E.coli. This was the first report on the MAb production of SsRV protein.