Development And Application Of PVS And PLRV ELISA Kit

Potato virus disease was a serious constraint to potato yield and quality. Serological method is important to detection and identification of plant virus. There are some defects that use virus particles as antigen to immunize rabbit and preparation of antiserum, such as low concentration of virus particle and difficult to purification. Used genetic engineering technology to express the fusion protein in vitro and used the fusion protein as antigen to prepare the polyclond antiserum. We can obtain polyclonal antibody with high specificity.In this study, the cp gene of potato virus S (PVS) and potato leaf roll virus (PLRV) were cloned and constructed its prokaryotic expression vector, and then expressed in E.coli (DE3), used fusion protein as antigen to prepare the polyclonal antiserum in rabbit respectively. The ELISA test kit of PVS and PLRV were used to detected samples of potato.First, the specific primers contain EcoR I and Hind Ⅲ restriction enzyme cutting sit of PVS and PLRV cp genes were designed, according to complete sequence of PVS and PLRV cp genes. The cp gene (885bp) of PVS and cp gene (627bp) of PLRV were amplified by RT-PCR, cloned to pET-30a and pET-28a for prokaryotic expression, the recombinant plasmids of pET-30a-PVS cp and pET-28a-PLRV cp were obtained.Second, transformed the plasmids of pET-30a-PVS cp and pET-28a-PLRV cp into BL21respectively, the fusion protein were expressed efficiently with IPTG inducing by SDS-PAGE detection, results show that the fusion protein of PVS and PLRV were expressed in high level and existed in the form of inclusion body. The expressed proteins were purified by Ni2+-NTA affinity chromatography and then dialyzed. The final concentration of PVS CP and PLRV CP were3.96mg/mL and2.72mg/mL, the purity were93%and97%respectively.Third, used the two expression proteines as antigen to immune the healthy rabbits, and obtained specific antiserum of PVS and PLRV, the optimal titer of PVS antiserum was1:51200; PLRV antiserum was1:25600respectively, Through sensitivity, specialization and Western blot test, results show that the two kinds of antiserum could reacted with PVS and PLRV respectively.Finally, Application the PVS and PLRV antiserum to assemble the potato virus disease detection kits, the ELISA test kit of PVS and PLRV were used to detected potato virus of species seedling and survey the distribution in Yunnan province, this study play an important role in potato disease control.