关键词: 乳酸克鲁维酵母, 凝乳酶, 分泌表达

Abstract: Objective:To construct Kluyvermyces lactis secreted expression vector and express recombinant prochymosin. Methods :The prochymosin gene encoding mature peptide was amplified by polymerase chain reaction, and then inserted into the downstream of the alpha-mating factor signal of the Kluyvermyces lactis expression vector pKLAC1. Recombinant plasmid pKLAC1-prochymosin was linearized by SacⅡand transformed into Kluyvermyces lactis strain GG799 with lithium chloride. Positive clones were chosen by YCB plates containing 5 mmol/L acetamide and the presence of insert was identified using PCR. Multi-copy integration can be detected using specifical integration primers. The positive transformants were fermented in flask and the proteins in the culture supernatant were deposited with TCA and assayed with Tricine SDS-PAGE. Results: It was proved that the fragment amplified is inserted into the Kluyvermyces lactis expression vector pKLAC1 correctly by PCR and gene sequencing. After lithium chloride transformation, the recombinant plasmid is integrated into the regions of homology within yeast genome. Chymosin activity of the medium is 83 U/ml after acid treatment. Tricine SDS-PAGE analysis proved that the molecular weight of chymosin is about 36 kD. Conclusion: Prochymosin Kluyvermyces lactis expression vector is successfully constructed and recombinant prochymosin is expressed.

Key words: Kluyvermyces lactis, chymosin, secreted expression