Cloning, Expression And Antigenicity Analysis Of A CDNA Encoding P46 KDa Antigen From Newborn 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. The adult worm and newborn larvae are the two invasive stages of Trichinella spiralis and they need some particular nosogenetic factors to invade the body of host. In this study the new born larvae (NBL) cDNA library of Trichinella spiralis was immunoscreened and antigen genes were cloned with the molecular biology and immunologic technology. This will lay the foundation to obtain diagnostic and protective antigen of Trichinellosis. NBL cDNA library of Trichinella spiralis was screened using the anti-sera from swine artificial infected with Trichinella spiralis. From 4.5×10~5 recombinant phages 156 positive clones were obtained and included that 36 new genes and 5 known genes. The positive clone pBK-CMV-WN10 encoding p46 kDa antigen of Trichinella spiralis contained cDNA transcript of 1352 bp in length with a full open reading frame (ORF) of 1218 bp which encoded 406 amino acids with molecular weight of 45 900 and isoeletric point of 5.43. The signal peptide sequence and N-glycosylation site indicated the putative peptide a secretory glycoprotein. The putative peptide consisted of two repeat domains of 138 amino acids with identity of 74% and a cystatin motif in the C-terminal which also showed 37% identity to the repeated domains and is different from cystatins of other nematodes. PCR of cDNAs from muscle larvae, newborn larvae, 3 day old adults and 5 day old adults confirmed the expression of this gene in all the stages of Trichinella spiralis. WN10 cDNA without signal peptide sequence was cloned into prokaryotic expression vector pET-28a and the recombinant plasmid pET-28a-WN10 was successfully constructed. The recombinant plasmid was transformed into BL21 (DE3) and induced by 1mM IPTG. The recombinant fusion proteins of 48 kDa were obtained by SDS-PAGE. The amount of the expressed proteins increased with time extending and reached 36.077 % of the total bacterial protein 4 hours after induction. To determine the immunogenicity of the recombinant proteins, WN10 recombinent protein and ES antigen of muscle larvae in T.s were analysised by mice sera which infected by T.s. The results showed that the antigenicity of recombinant protein was equal to ES antigen of muscle larvae in T.s and could be used to diagnose the infection of T.s, but the specificity of WN10 recombinent protein should be determined furthermore. Western-blotting showed the recombinant proteins were recognized by positive sera from swine and mice infected with T.s. WN10 recombinant protein was also used to detect antibody of mice immunized and infected with T.s by indirect ELISA, the results showed the...