Construction And Expression Of The Recombinant Plasmid PPIC9K-pZP3α-hCGβ－CTP～(109～145) In The Yeast Pichia Pastoris

Aim: For the purpose of making the contraception vaccine pZP3 a -hCG P-CTP109-145 which has multiple specific epitopes, use gene engineering technology to recombinant plasimid pPIC9K-pZP3 a -hCG P-CTP109-145 and express this plasimid in methylotrophic yeast Pichia pastoris. Methods: The two pairs of designed primers were based on pZP3 a and hCG P-CTP109-145 cDNA sequences. The pZP3 a -hCG P -CTP109-145 chimera was amplifiled by overlapping PCR. The chimera was cloned into pPIC9K plasmid and transformed into E.coli DH5 a . The recombinant pZP3 a -hCG P -CTP 109-145 was testified its DNA sequence and orientation by PCR and DNA sequencer. The recombinant plasimid pPIC9K-pZP3 a -hCG P-CTP109-145 was transformed into electroporated Pichia pastoris and screened the positive strains on MD plates without histidine. Multi-copy recombinants were screened on the G418-YPD plates and then were incubated with the supernatant containing methanol.The recombinant pZP3 a -hCG P -CTP 109-145 protein were secreted in the supernatant and were verified. Resu I ts: pZP3 a -hCG P -CTP10945 was constructed by three steps of PCR and was totally inserted into vector plasmid pPIC9K with pZP3 a DNA and hCG P -CTP 109-145DNA linked together. The result of the sequencer was entirely consistent with expectation. The protein secreted by multi-copy recombinants screened on the 4mg/mL G418 plate were evaluated by SDS-PAGE and Western blotting. Proteins of expression in multi-copy recombinant were able to react with monkey-anti-pZP3 a antibody and rabbit-anti-hCG antibody specifically. Cone I us i on: Recombinant pZP3 a hCG P-CTP109-145 was constructed and expressed in the yeast Pichia pastoris successfully. It provides a foundation for further contraception vaccine research.