Expression Of Phosphoprotein P2 Of Cysticercus Cellulosae In Pichia Pastoris System

Acid ribosomal phosphoprotein(P2 protein) of Cysticercus cellulosae locates at the60S subunit of ribosome. It was proved that the P2 protein plays an essential role inprotein synthsis via subunit deletion nd suPression experiments. It was reported that theP2 protein of Cysticercus cellulOsae can be distinguished by the serum of theneurocysticercosis. So, it focuses on if the P2 protein could be used as antigen indiagnosis and prevention and how enough P2 protein could be obtained. In the thesis,Pichia Expression System was used to express recombinant P2 protein in viho. First, theinterested P2 gene was got by digesting the pGEM-P2 vector using restrictionendonuclease,then the inierested P2 gene was inserted into the secreted expression vectorpPIC9K and transformed into E.coli.. Positive recombinants were selected. sequencedand named pPIC9K-P2 .It was linearized by Sal I and BgII,then the linear DNAtransfored iflto Pichia Pastoris GSll5 by electroporation. The expression vectorpPIC9K-P2 integrated into GSll5 via homologous recombinantion betWeen thetransforming DNA and regions of homology within the genome. TWo differentphenotypes were generated --- HIS+ MUT+ (Methanol utilization plus) and HIS+MUTs(Methanol utilization slow).MD was used to select HIS+, MD and MM were used todistinguish MUr and MUTs,and the in vivo method utilizes hyper-resistance to G4l8 -to screen fOr possible multicopy inserts. Analysis of PCR showed that the interested P2'gene was integrated within the genome of the multicopy recombinans, and the multicopyrecombinants were named GSll5/pPIC9K-P2HIS+MUT+ and GS1l5/pPIC9K-P2HIS+MUTs. Test expression of both HIS+MUT+ and HIS+MUTs were induced withmethanol and taken time points, then the secreted supernatant was tested by SDS-PAGEand Westem-blot. The results showed that the P2 protein has been expressed successfullyin Pichia Pastoris.The expression protein was about l2.6kD and can be distinguished bythe positive serum of Neuro-cysticercosis.