Cloning And Expression Of Taenia Solium 45W Gene In Pichia Pastoris System

Taenia solium 45WB2 protein is a membrane combined protein of oncosphere and plays an essential role in penetrating and forming cyst in pig tissues. It has been proved that the infection of T·solium can be prevented effeciently by injecting T·ovis 45W recombinant vaccine. This study is to express large amounts of 45WB2 protein in vitro using the P·pastoris expression system, to solve the difficulty of the limitation of immunogical antigen resources. Total RNA was extracted from oncosphere hatched and activated, the 45WB2 gene specific primers were designed using Oligo software. A 459 bp specific fragment was amplified by RT-PCR and ligated into pGEM vector. It was identified by restriction endonuclease analysis, PCR and sequencing that the fragment contained the complete open reading frame (ORF) of the 45WB2 gene. The homologies of the nucleotide sequence and amino acid sequence were 94.8% and 95.4% respectively compared with the sequence published in Genbank. The recombinant plasmid pGEM-45WB2 was digested with EcoRI and NotI, releasing the 45WB2 gene fragment. The fragment subsequently was inserted into the EcoRI and NotI sites of the secrected expression vector pPIC9K and was transformed into E. coli JM109. Positive recombinants named pPIC9K-45WB2 was linearized by SalI, then the linearized DNA was transformed into P·pastoris SMD1168 by electroporation. MD was used to select HIS+ transformants and then utilized hyper-resistance to G418 to screen for possible multicopy strains. PCR analysis showed that the gene of interest was integrated within the genome of the mulicopy recombinants. The recombinants were induced to express products by 1% methanol. The cultures supernatant was collected and tested by SDS-PAGE and Western-blot. It showed that the 45WB2 gene was expressed successfully in P·pastoris and the molecular weight of the expressed protein was about 16 ku. The 16 ku protein can be recognized by the positive serum from patient cysticercosis.