Cloning and prokaryotic expression of serine proteinase inhibitor gene of Trichinella spiralis

A pair of specific primers derived from Trichinella spiralis serine proteinase inhibitor gene(Tsserpin) in GenBank was designed and used to amplify Tsserpin gene by RT-PCR from total RNA extracted from T.spiralis muscle larvae.The RT-PCR product was purified,digested and ligated into the expression vector pET30a and identified by PCR,restriction digestion and sequencing after transformation into Escherichia coli DH5α.pET30a-Tsserpin plasmid was transformed into E.coli BL21(DE3) and induced by IPTG.The expressed product was analyzed by SDS-PAGE and the antigenicity of the purified recombinant protein was identified by Western-blot with T.spiralis-infected swine serum.The EcoRⅠ+XhoⅠ digestion,PCR amplification and sequencing analysis demonstrated that the constructed pET30a-Tsserpin contained a insert of 1122bp encoding 373 amino acids,which shared 99% identity to Tsserpin gene in GenBank.The recombinant protein was highly expressed in the form of inclusion bodies with the molecular weight of about 48.5ku.Western-blot analysis indicated that the purified recombinant protein could be specifically recognized by T.spiralis-infected swine serum with a good antigenicity and might be a candidate antigen for the immunodiagnosis of swine trichinellosis.