Cloning, Expression And Characterization Of WR29 Antigen From Muscle Larvae Of Trichinella Pseadospiralis

As a zoonotic parasitic disease, trichinellosis shows a worldwide distribution and has a high prevalence in China, could infect over 150 mammals besides human, not only lead to enormously economic loss of husbandry and meat industry but also severely threat to public health. Trichinella spiralis muscle larvae is infected with host,On the basis of judge outcome from bionomics and molecular biology, Trichinella is distinguished two main categorves, it is at host musculature moderate energy encyst to number, it is at myocyte middle not encyst to tother genera. Trichinella pseadospiralis is one of species of Trichinella that abroad parasitizes vertebrate skeletal muscle cell without encystment in larvae stray time,the span of the even pathology injure came down to myocyte.experience four exuviations to develop into imago in 30h.Consequencely,it gave birth to newborn larvae in 96h and newborn larvae invaded into all skeletal muscle by lymph and blood. Finally, newborn larvae grew up infective muscle larvae, which could survival in animal and human body for long term. Muscle larvae take in host nutrition and endangered to host health. The multiplicity Trichinella spiralis antigens made a difficulty for a large-scale reproducing in vitro,immunodiagnosis and prevention .Recently, Trichinella spiralis study only focus on antigen , but its antigen is multiplicity. Though ES was used of immunodetection in trichiniasis ideally, however, epitope of ES had nonspecific reaction with factor of human serum. Meanwhile, it could lead to a false positive diagnosis and there were difficulty for large- scale preparation. In this study the muscle larvae (ML) antigen gene WR29 of Trichinella pseadospiralis 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, WR29 cDNA gene was analysis after knowing it's sequence. WR29 antigen of Trichinella pseadospiralis contained cDNA transcript of 961bp in length,from 3 to 734 is coding region which encoded 243 amino acids with molecular weight of 27 879 and isoelectric point of 5.34. The contents of GC is 43.91% .WR29 cDNA without signal peptide sequence was cloned into prokaryotic expression vector pET-28a and the recombinant plasmid pET-28a-WR29 was successfully constructed. The recombinant plasmid was transformed into BL21 (DE3) and induced by 1mM IPTG. The recombinant fusion proteins of 27.9kDa were obtained by SDS-PAGE. The amount of the expressed proteins increased with time extending and reached 58.1% of the total bacterial protein 4 hours after induction.To determine the reactionogenicity of the recombinant proteins, recombinent protein of muscle larvae in T.p 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 Proteasome activator subunit are known to provoke a strong immune response, so we presumed that the Proteasome activator subunit could be used to diagnose the infection by T.p. But the specificity of WR29 recombinent protein should be determined furthermore. WR29cDNA was reverse-transcribed from total RNA. of muscle larvae, newborn larvae, 3 days old adults and 5 days old adults confirmed the expression of this gene in all the stages of Trichinella pseadospiralisThe stronger reactive signal and high copy cDNA encoded proteasome activator subunit will probably become a candidate gene for the T.pseuodospiralis diagnosis antigen. These results play a foundation part in the further study of the genic recombinant antigen of T.pseuodospiralis.In this study,it promoted antigen of Trichinella pseadospiralis muscle larva to become candidate gene, antigen gene recombination in Trichinella pseadospiralis every growth stage, expression of polygenic recombination antigen and make foundation for establishment of sensitive and specific immunology detect method.