Cloning and prokaryotic expression of ASP gene from Angiostrongylus cantonensis.

The purpose of this study was to clone and express the complete open reading frame of ASP gene from Angiostrongylus cantonensis and analyze the immunogenicity of the recombinant protein. The coding region of ASP gene was amplified from the larvae cDNA plasmid library by PCR. The PCR product was then cloned into the prokaryotic expression vector pET-30a (+) and transformed into E.coli BL21 (DE3). After that,the IPTG induced product was purified by Ni-IDA affinity chromatography,then detected by SDS-PAGE and identified its immunogenicity by Western blotting. Results showed that the coding sequence of ASP gene contains 366 base pairs encoding 121 amino acids,with a theoretical molecular weight of 13398.26 Da. PCR,double enzyme digestion and DNA sequencing assay confirmed that the recombinant plasmid pET-30a (+)-ASP was constructed successfully. By IPTG inducing,the soluble expression of recombinant protein was obtained in E.coli BL21. The affinity chromatography purified recombinant protein which could be recognized by the serum of the angiostrongyliasis cantonensis patients with Western-blotting. It's concluded that the ASP gene has been efficiently expressed in prokaryotic expression system with immunogenicity in vitro.