水稻草状矮缩病毒P2基因多克隆抗体的制备及应用

为了进行水稻草状矮缩病毒(Rice grassy stunt virus, RGSV)诊断方法和蛋白功能研究。通过RT-PCR方法从感染RGSV的水稻中克隆该病毒的P2基因，并将此基因片段重组到原核表达载体pDEST17上。将重组载体转化E. coli Rosetta，经IPTG诱导后获得分子量约为29 kDa含HIS标签的融合蛋白。以诱导的融合蛋白为抗原，免疫新西兰大耳白兔获得多克隆抗体，经酶联免疫检测发现其效价达到l:8192。并用制备的抗体建立了特异、灵敏的检测RGSV的IC-RT-PCR和Dot-blot ELISA方法，为该病毒的检测、诊断提供了保障，同时也为P2蛋白的结构和功能研究奠定了基础。

Prokaryotic Expression of P2 protein of Rice Grassy Stunt Virus, and Preparation and Application of Its Polyclonal Antibody

P2 gene of Rice grassy stunt virus was amplified by RT-PCR from the infected rice leaves, and it was subcloned into prokaryote expression vector pDEST17 to construct recombinant expression plasmid, which then was transformed into E. coil Rosetta. The 29 kDa HIS6-tag fusion protein was obtained with induction of IPTG from E. coil Rosetta cells, which was used to immunize the healthy rabbits as an antigen. The antiserum of P2 protein was obtained after five times injections and the ELISA analyses showed that the potency of antiserum was 1：8192. Then, the antiserum was used to establish the Immunocapture RT-PCR and Dot-blot ELISA method to detect the RGSV. Therefore, the P2 protein and antiserum provided the technical support for the diagnosis of RGSV disease and protein function study.