Clonging, Expression And Identification Of P53 Antigen Gene Of Trichinella Spiralis

Trichinellosis caused by infection of Trichinella spp is a world wide distributed parasitic zoonosis and harms to human health and livestock development. Muscle larvae excretory-secretory (ES) antigen was used in immunodiagnosis of trichinellosis and showed good sensitivity and specificity. However, it is difficult to produce the native antigen in mass and standardized. Application of genetic engineering technology to produce recombinant protein as antigen is expected to solve this problem. TsP53 protein is one of the major antigenic components of the muscle larvae ES antigens. p53 gene fragment was amplified by RT-PCR from the total RNA of Trichinella spiralis muscle larvae and cloned into the expression vector pET30a. After transformation to E. coli DH5αand identification by PCR, restriction enzyme digestion and sequencing, pET30a-TsP53 plasmid was transformed to E. coli BL21 (DE3) and induced by IPTG for expression. The expressed product was analyzed by SDS-PAGE and the antigenicity of the purified recombinant protein was identified by indirect ELISA with T. spiralis infected swine serum. Mice were immunized with 20μg TsP53 recombinant protein for 3 times and challenged with 200 infective larvae per mouse one week after the third immunization. Recovery of 7 days old adult worms, production of newborn larvae of female adult in vitro and muscle larvae on 35 day post infection were counted and the anti-TsP53 IgG level were detected by ELISA.It was demonstrated that the recombinant pET30a-TsP53 plasmid contained an insert of 1176 bp encoding 391 amino acids which was 99% homologous to T. spiralis p53 gene (U25127)in GenBank database. The recombinant protein was highly expressed in the form of inclusion body with the molecular weight of about 50 ku. The purified recombinant protein could be specifically recognized by T. spiralis infected swine serum. The reduction rate of 7 days old adult worms, newborn larvae and muscle larvae were 14.34%, 16.93% and 38.68%, respectively. It is suggested that TsP53 recombinant antigen could induce a high immune protection in mice against T. spiralis. The anti-TsP53 IgG level in immunized mice was significantly higher than that in control. This will lay the foundation for immunodiagnosis and vaccine development of trichinellosis.