Construction And Expression Of The Recombinant Plasmid β HCG-pPIC9K In The Yeast

To construct a recombinant plasmid β-hCG-pPIC9K and transform it into methylotrophie yeast Pichia pastoris for expressing β-hCG protein with the intention to develope contraceptive vaccine by using β-hCG as in imunogen. β-hCG DNA fragment with EcoR I and Not I at 5'and 3'termini was prepared by PCR using plasrnid β-hCG--PBS kS as template and a pair of designed primers which carried EcoR I in upstream and Not I in downstream. After digested by both EcoR I and Not I, β-hCG DNA fragment was linked with vector plasmid pPIC9K by T4 DNA ligase. The recombinant was transformed into competent DH5. Positive clones were screened. The combination plasmid β-hCG-pPJC9K isolated and purified from positive clones was comfirmed by PCR, digesting with both EcoR I and Not I, and sequensing. All the results showed that β-hCG DNA was linked with vector plasmid pPIC9K successfully. The transformant was transformed into electroporated Pichia pastoris and screened the positive strains on MD plates without histidine. Multi-copy recombinants were screened on the G4 I 8-YPD plates. Concerning the expressing process, we formulated an improved condition to increase expression of the target gene. We found that the expression was much improved in the methylenic concentration of 0.5% after 4 days. The expressed protein has been evaluated by SDS-PAGE and Westernblot,and demonstrated the protein being reacted with rabbit-anti-hCG. All the results showed that the recombinant plasrnid β-hCG-pPIC9K was constructed and expressed in the yeast Pichia pastoris successfully.