Prokaryotic Expression Of PPro2Glu And Cp Genes Encoded By Strawberry Mottle Virus

Strawberry mottle virus(SMo V)is a two-component RNA virus with glutamate protease(Pro2Glu)and coat protein genes(CP)located on RNA2.In this study,RT-PCR technology was used to detect SMo V,and specific primers were designed to clone Pro2 Glu gene and cp gene.After sequencing,sequence alignment was performed and a phylogenetic tree was constructed.The Pro2 Glud gene and the cp gene were inserted into the prokaryotic expression vector p ET32 a,and the recombinant vectors p ET32a-Pro2 Glud and p ET32a-CP were respectively transformed into E.coli.After induction by IPTG and affinity purification with Ni2+-NTA,SDS-PAGE and Western-blot analysis showed that they were successfully obtained.Pro2 Glud fusion protein and CP fusion protein,to lay the foundation for the preparation of SMo V specific antiserum.1.Cloning and sequence analysis of SMo V Pro2 Glu gene and cp gene41 strawberry samples were collected from Guohe Town,Lujiang County,Hefei City,total RNA was extracted,and the first strand of c DNA was reverse transcribed.Design specific primers to amplify SMo V Pro2 Glu and cp genes,clone and sequence.The Pro2 Glu and cp genes are 1842 nts and 1947 nts,respectively,and the encoded proteins are 613 and 649,respectively.Sequence analysis showed that the nucleotide sequence of the Pro2 Glu gene of the SMo V Anhui isolate(SMo V-AHHF)has a high similarity with the sequence of the Chinese isolates of SMo V,reaching 89.7%-97.8%,while the sequence similarity with the foreign isolates of SMo V is low,71.0%-93.5%,of which the sequence similarity with the two Chinese isolates of SMo V(SMo V-SDHY5)and(SMo V-BJMX7)is the highest,up to 97.8%,and the sequence similarity with the Dutch isolate(SMo V-1134)is the lowest,Which is 71.0%.The nucleotide sequence of the cp gene of SMo V-AHHF has a high similarity with the sequence of each isolate of SMo V in China,reaching 97.7%-98.6%,while the similarity with the sequence of each foreign isolate of SMo V is low,ranging from 77.5% to 94.7%.The Chinese isolate(SMo V-DGHY26-2)has the highest sequence similarity at 98.6%,and the Czech Republic isolate(SMo V-A)has the lowest sequence similarity at 77.5%.It shows that the nucleotide sequence variation of Pro2 Glu and cp gene of the Chinese isolate of SMo V is relatively small.According to the alignment results of the SMo V-AHHF Pro2 Glu sequence and the SMo V-AHHF CP sequence with the sequence of each isolate of SMo V,a phylogenetic tree was constructed.In the constructed Pro2 Glu phylogenetic tree,SMo V Dutch isolate 1134 forms a large group,and the rest of SMo V isolates form a large group.Among them,Hefei isolate Pro2 Glu from Anhui,all SMo V Chinese isolates and Czech Republic isolate C form a group.In the constructed CP phylogenetic tree,the SMo V Czech Republic isolate A alone formed a large group,and the remaining SMo V isolates formed a large group,of which the Anhui Hefei isolate CP and all SMo V Chinese isolates formed a group.These results indicate that the genetic variation between SMo V-AHHF Pro2 Glu and SMo V-AHHF CP and SMo V Chinese isolates is relatively close,and the genetic variation between Czech isolates is high.2.Prokaryotic expression of SMo V Pro2 Glu and cp genesThe SMo V Chinese isolate Pro2 Glu and cp genes were amplified by PCR technology,and the genes were cloned into the prokaryotic expression vector p ET32 a to obtain recombinant plasmids p ET32a-Pro2 Glud and p ET32a-CP.The candidates were transformed into Escherichia coli,the final concentration was 0.1 mmol/L-1 IPTG induced expression and Ni2+-NTA affinity column purification.SDS-PAGE and Western blot analysis showed that His monoclonal antibody can react specifically with the fusion protein purified in E.coli,thereby obtaining p ET32a-Pro2 Glud with a molecular mass of about 85 k Da and p ET32a-CP fusion protein with 90 k Da.