The Cloning And Prokaryotic Expression For Coat Protein Gene Of Chinese Isolate Of Grapevine Leafroll Associated Virus 2

China is abundant in grapevine and its grapevine product is the fifth in the world .In recent years, grapevine leafroll disease conduced by grapevine leafroll associated virus (GLRaV) occurred in many countries and normally leaded the yield losses as much as 20% to 40%.Currently, there are at least nine distinct GLRaVs .Among these, GLRaV-1, 2 and 3 are the most frequently detected in leafroll-infected grapevines. There had been some associated report about GLRaV-3 but no report about GLRaV-2 in China. In this study many grapevine varieties were detected for GLRaV-2 by DAS-ELISA and cloned to analyze the sequence of the CP gene and express the CP gene in E. coli.There were 5 grapevine varieties were confirmed them infected GLRaV-2 through detection by DAS-ELISA from 17 grapevine varieties. Viral dsRNA was extracted from a GLRaV-2-infected grapevine variety "Cabernet France" .The cDNA contained the CP gene of GLRaV-2 about 673bp was amplified by RT-PCR with specific primers and then was inserted into clone vector pMD18-T. The recombinant plasmid named GLRaV-2-sh was choosed for sequencing from 5 plasmids contained the CP gene of GLRaV-2. The sequence of GLRaV-2-sh-CP was logged on GenBan(AY842932) after sequencing. The result of sequence analysis indicated that this gene was all 98% and 99% identical to GLRaV-2-pn-CP and GLRaV-2-se-CP at nucleotide and amino acid level, respectively. This gene was 94% identical to GLRaV-2-H4-CP at amino acid level.The CP gene was inserted into prokaryotic expression vector PET-22b(+) and expressed in E. coli BL21(DE3) strain. The product was identified by SDS-PAGE and the result showed that the specific expression of 22K Da protein was achieved. Western blot analysis was carried out with expression product and virus-specific monoclonal antibody to GLRaV-2. The resulte of western bolt confirmed that the prokaryotic expressed protein can react with the antiserum.