毕赤酵母表达杀菌/渗透性增强蛋白N端片段

目的 探讨在毕赤酵母中进行杀菌／渗透性增强蛋白N端片段的分泌表达。方法从质粒pUCm-BPI上PCR扩增得到编码BPI氨基端196个氨基酸的基因片段(BPl588)。将该基因克隆到酵母表达载体Ppic9K上，使其准确融合于α交配因子分泌信号之后，电击转化毕赤酵母宿主菌GS115／His-。对酵母重组子的基因组DNA作PCR和总RNA作RT-PCR．分析。筛选出的转化子用甲醇作诱导物在29℃进行诱导后，分泌到培养基中的表达产物用于革兰氏阴性细菌E.coli JM109的抑菌实验。结果BP1588基因已整合到毕赤酵母染色体基因组中，并有转录产物mRNA的存在，表达产物能够抑制革兰氏阴性细菌的生长。结论利用毕赤酵母进行杀菌／渗透性增强蛋白N端片段的分泌表达是可行的。

Expression in P. pastoris of N-terminal fragment of human bactericidal/permeability-increasing protein

[Objective] To express N-terminal fragment in P.pastoris. [Methods] Using PCR, cloning and protein expression techniques, a 588 bp DNA fragment encoding for the N-terminal fragment of human bactericidal/permeability-increasing protein (BPI) was subcloned into P.pastoris vector Ppic9 K and then transformed into P.pastoris strain GS115 to get a recombinant protein corresponding to the 4-199 amino acids of human BPI, tested its bactericidal activity to E.coli JM109. [Results] The BPI588 gene was integrated into the pichia genome and was transformed into mRNA; cell-free supernatant showed bactericidal activity. [Conclusion] P.pastoris can be a potential host strain for the production of N-teminal fragment of BPI.