Prokaryotic expression of lumpy skin disease virus ORF141 protein and preparation of polyclonal antibody
In order to obtain the ORF141 protein of lumpy skin disease virus(LSDV)and its polyclonal antibody,primers were designed using SnapGene software referring to the LSDV ORF141 gene sequence from GenBank,and ORF141 gene was amplified by PCR using the LSDV genome as template,and the prokaryotic expression recombinant plasmid pET-30a(+)-ORF141 was constructed.Transformed into Rosetta E.coli-expressing cells,expression was induced with isopropylthiogalactoside(IPTG),and the interest protein was identified using SDS-PAGE and Western-blot.To prepare polyclonal antibodies,New Zealand white rabbits were immunized with expressed protein and the properties of polyclonal antibodies was deter-mined by immunofluorescence assays.The results showed that the pET-30a(+)-ORF141 prokaryotic expression vector was successfully constructed,and the target protein was induced to express at 1 mmol/L IPTG at 37 ℃,with the size of about 25 ku,which mainly existed in the form of inclusion bodies.The titer of the prepared anti-LSDV ORF141 polyclonal antibody could reach 1∶409 600,and the immunofluorescence test showed that it could recognize the natural protein encoded by ORF141 in LSDV virus,which lays a founda-tion for further research on the biological function of the protein encoded by LSDV ORF141 gene.
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