Prokaryotic Expression And Polyclonal Antibody Preparation Of Porcine ISG43 And ISG60 Proteins

ISG60,a member of the IFIT family genes,is a class interferon inducible genes that are highly conserved in evolution.The ISG60 gene was highly expressed under the stimulus of interferon,virus and lipopolysaccharide,a major role in the initiation of translation,the replication of virus,the feedback regulation of type I interferon.ISG43 was first discovered and reported its ubiquitination function by Liu in 1999,also known as UBP43,and it is a member of the ubiquitin specific protease(UBP)family member.In 2002,Malakhov discovered the ISGylation function of UBP43,and that UBP43 gene with partial deletion will lead to apoptosis because it occurs interferon allergy.Thus ISG43 plays a key function to maintain the dynamic balance of ISG.In this study,ISG60 and ISG43 genes were cloned,and both genes are connected to pMD18-T vector.Insertion of PET-32a(+)cloning site by Xho I and BamH I restriction enzyme digestion after correct sequencing,the recombinant prokaryotic expression plasmid of PET-ISG60 and PET-ISG43 were constructed successfully.The recombinant plasmid was transformed into Rosetta(DE3)and induced target gene expression with different concentrations of IPTG,analysis by SDS-PAGE and Western-Blot.It is shown that ISG43 showed the best expression under condition of 0.5 mM IPTG,ISG60 showed the best expression with 0.05 mM IPTG.Both ISG43 and ISG60 could be expressed in two forms:inclusion body and soluble protein.Purification of both proteins using soluble protein form obtained an active immunogen,and the purified recombinant ISG43 and ISG60 active proteins were used as antigens to immunize mice with Freund’s adjuvant.After four times of immunization program,the polyclonal antibodies with high reactivity and specificity were obtained.And the reactivity of the poly clonal antibody with prokaryotic expression protein,or the reactivity of the poly clonal antibody with corresponding protein in PK-15 cells were detected by Western blot.As the results shown,both poly clonal antibodies could well reacted with prokaryotic expression protein at the dilution of 1:2000,1:4000,1:8000.Similarly,both poly clonal antibodies could well reacted with the corresponding protein expressed in PK-15 cells by polyI:C under the dilution of 1:600,1:1200.