Construction Of Recombinant Capripox Virus System For Expression Of Foreign Proteins

Goat pox virus belongs to poxviridae,which includes goat pox virus,sheep pox virus and lumpy skin disease virus,cause serious diseases of goat,sheep and cattle respectively.Its typical clinical symptoms are papules,blisters,pustules,nodules and scabs on animal skin,with a high mortality rate to young animals.The virulence of sheep pox virus isolate was weakened by continuous passage of chicken embryo,and the immune effect of attenuated vaccine was very good.Due to the large genome of poxvirus,it can accommodate a large number of foreign genes without affecting its normal replication.Therefore,it is possible to take the poxvirus vaccine strain as the carrier,load the protective genes of other pathogens,and construct a monovalent or multivalent recombinant vaccine to achieve one shot to prevent multiple diseases.However,the construction process is more complex.In order to make better use of the poxvirus vaccine strain to construct a multivalent vaccine,In this study,an expression system of sheep pox virus for expressing foreign genes was constructed.In this study,pG-klp2-VV7.5-GFP plasmid,pUC18-p11-gpt-VV7.5 plasmid and p UC18-I1L-Lacz-VV7.5 plasmid are used as templates respectively.VV7.5-GFP-loxp,p11-MCS(multiple cloning site)-loxp-gpt-VV7.5 and I1L-MCS-loxp-Lacz-VV7.5 were amplified by ordinary PCR.After that,the p11-MCS-loxp-gpt-VV7.5 and I1L-MCS-loxp-Lacz-VV7.5 gene fragments were digested by Bsi WI and ligated into the Acc65 I site of the p GM-TK13 vector to construct the two plasmids,and then VV7.5-gfp-loxp gene fragment was connected to the Bsu36 I site at the 3'end of the new fragment in the constructed vector.After successful construction was identified,plasmids were extracted,Lamb testicular cells that had been infected with the poxvirus weakly virulent vaccine strain GTPV AV41 were transfected with Lipofectamine 2000 and examined under a fluorescence microscope every 12 h,After 36 to 48 h of fixation with low melting agarose,fluorescent spots were picked and stored in DMED and were freeze thawed repeatedly three times at-20? to 37? and 10 fold dilutions were used to replate lambs testicular cells.The fluorescent spots were then microscopically observed,picked,diluted,and seeded so repeatedly for 4~6 rounds until all the cells were fully fluorescent,and their genomes were extracted for PCR identification.The VV7.5-gfp-loxp,p11-MCS-loxp-gpt-VV7.5 and I1L-MCS-loxp-Lacz-VV7.5 gene fragments were constructed into the vector to obtain the recombinant transfer plasmid p GM-TK13-p11-MCS-loxp-gpt-VV7.5 and p GM-TK13-I1L-MCS-loxp-Lacz-VV7.5.After transfection of the two recombinant plasmids into lamb testicular cells that had been previously inoculated with the GTPV AV41 vaccine strain,The cells expressing GFP gene,namely green fluorescence,were produced.After picking out the fluorescence and subculturing the offspring virus suspension for 5 times,the purified recombinant virus p GM-TK13-p11-MCS-loxp-gpt-VV7.5 was identified by PCR.