Cloning, Expression And Characterization Of WM5 Antigen From Muscle Larvae Of Trichinella Spiralis

Trichinellosis is a world wide distributed zoonotic parasitic disease caused by nematode Trichinella and has a high prevalence in China. More than 150 mammals besides human could be infected and this disease could not only lead to enormously economic loss of husbandry and meat industry but also severely threat to the health of human. The complexity of Trichinella spiralis antigens and difficulties in reproducing in a large number in vitro made the immunodiagnosis and prevention of Trichinellosis difficult. In this study the muscle larvae (ML) antigen gene WM5 of Trichinella spiralis was cloned with the molecular biology technology and was detected using immunologic technology. This will lay the foundation to obtain diagnostic and protective antigen of Trichinellosis.In this study, WM5 cDNA gene was analysis after knowing it's sequence. WM5 antigen of Trichinella spiralis contained cDNA transcript of 1315 bp in length with a full open reading frame (ORF) of 1122 bp which encoded 373 amino acids with molecular weight of 42 000 and isoelectric point of 5.22. The signal peptide sequence site indicated the putative peptide a secretory glycoprotein.WM5 cDNA without signal peptide sequence was cloned into prokaryotic expression vector pET-28a and the recombinant plasmid pET-28a-WM5 was successfully constructed. The recombinant plasmid was transformed into BL21 (DE3) and induced by 1mM IPTG. The recombinant fusion proteins of 37.7 kDa wereobtained by SDS-PAGE. The amount of the expressed proteins increased with time extending and reached 51.1% of the total bacterial protein 4 hours after induction.To determine the reactionogenicity of the recombinant proteins, recombinent protein of muscle larvae in T.s were purified and immunized to rabbits. The results showed that the recombinant protein was very strong. By NCBI BLAST, we found the recombinant protein was a serpin, and proteinase inhibitors are known to provoke a strong immune response, so we presumed that the serpin could be used to diagnose the infection by T.s. But the specificity of WM5 recombinent protein should be determined furthermore.The predicted amino acid sequence of the clone had an identity of only 30% to the serine proteinase inhibitors (serpins) from Caenorhabditis elegans or Brugia malayi. At the putative reactive region, however, the identity was about 50%. This research will lay the foundation for the immune dignosis and control of trichinellosis.