LMoV CP原核表达 抗血清制备及绿体中的检测.docx

LMoV LMoV CP原核表达、抗血清制备及叶绿体中的检测 Prokaryotic Expression，Antiserum Preparation and Detection in Chloroplasts of LMoV CP Abstract 三砂mottle virus(LMoV)iS belonged to Potyvirus，which iS one of the most common viruses infecting lily．LMoV Call cause mottled or striped symptom after infecting lily． Moreover,LMoV can reduce the production and quality of lily bulbs．At present,the pathogenesis of LMoV is not clear．the research of proteins function which encoded by LMoV iS stin blank．which seriously hinders the prevention and treatment of lily virus diseases．In this paper，a serologieal method for detection of LMoV Was established，and the effect of LMoV on lily chloroplasts Was preliminarily proved．which lays a foundation for elucidating the pathogenesis of LMoV． According to the sequence of LMoV gene(HM22252 1．1)reported on GenBank，we designed the primers and amplified the coat gene of LMoV from the total RN A of lily leaves bv RT．PCR and obstained about 822 bp DNA fragment．The fragment Was cloned to the cloning vector pMDTM 1 8．T．The recombinant plasmid pMDTMl 8．CP was transformed imo Escherichia coli DH5伍．and then was sequenced after identified bV PCR and restfiction enzymes．nle results showed that we had successfully constructed recombinant plasmid pMDrMl8．CP． We amplified the coat gene of LMoV from the recombinant plasmid pMDIMl 8-CP by PCR，then the gene fragment was cloned to the expression vector pET-28“+)．The recombinant plasmid pET28a-CP was transformed into Escherichia coli BL2 1(DE3)and Was induced by IPTG．nle expression products were analyzed by SDS．11le results showed that approximately a 34 kDa recombinant protein witll His tag was expressed．The expression yield of recombinant protein Was increased by optimizing induction temperature and time． The result showed that tlle optimal expression condition of recombinant protein was 30℃，4 h． We purified the recombinant protein by Ni—IDA．Sefiuose under denaturing conditions and obtained a single recombinant protein band wi