Expression In Pichia Pastoris System And Location Study Of SLC10-An Unknown Gene From Taenia Solium

An unknown gene, SLC10, was cloned by spliced leader-based PCR from Taenia solium. The ORF of SLC10 was found to be 507bp, encoding an 18.2kD protein. The genomic DNA of SLC10 was found to be 1,107bp consisted of two exons and one intron. This study is to express SLC10 protein in vitro using the P-pastoris expression system and understand its function by Location Study. Study its protein not only to help us to understand its biotic characters but also to identificate vaccine or diagnostic candidates. Total RNA was extracted from oncosphere hatched and activated the SLC10 gene specific primers were designed using DNAStar software. A 507 bp specific fragment was amplified by RT-PCR. The products was digested with EcoRⅠand NotⅠ, releasing the SLC10 gene fragment. The fragment subsequently was inserted into the EcoRⅠand NotⅠsites of the secrected expression vector pPIC9K and was transformed into E. coli JM109. Positive recombinants named pPIC9K-SLC10 was linearized by SacⅠ, 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. It showed that the SLC10 gene was expressed successfully in P·pastoris and the molecular weight of the expressed protein was about 18 ku. The 18 ku protein can not be recognized by the positive serum from patient cysticercosis. ELISA results showed that all detected sera, including 70 normal and 75 cysticercosis sera, did not react with purified recombinant SLC10 protein. Using an immunohistochemical method, it was proved that the native SLC10 protein distributed mainly in inner cyst walls but not in the scolex in C. cellulosae. Together with predicted results, it is suggested that the SLC10 protein is a non-secretory structural protein, not involved in induction of the host's immune reactions against infection at least at the larval stage.