【摘要】 目的:在毕赤酵母SMD1168中高效表达抗菌肽PR39,并检测其抗菌活性。方法:根据毕赤酵母密码子偏爱性,人工合成4条寡核苷酸片段,通过重叠PCR获得PR39核苷酸序列,将其插入酵母表达载体pPIC9K,重组表达质粒pPIC9K-PR39经PCR敲除PR39 5’端多余的长度为24bp的核苷酸序列得到pPIC9K-PR39-D,再经PCR、酶切连接构建5’UTR已删除8个核苷酸GGATCCAA的新型毕赤酵母表达载体pPIC9K-PR39-D-E;pPIC9K-PR39-D-E转化到毕赤酵母SMD1168,经PCR鉴定及G418筛选,用0.5%的甲醇诱导表达,Tricine-SDS-PAGE分析,再利用琼脂糖扩散法测定发酵上清的抗菌活性,表达产物经反相层析及SP-Sepharose层析柱纯化测定其表达量。结果:经Tricine-SDS-PAGE检测,在4.7kDa左右处可见目的蛋白表达条带,纯化后测定蛋白浓度表达量约为175.6mg/L。获得的抗菌肽PR39对E.coli DH5α,有明显抑菌活性。结论:经改建后新型毕赤酵母表达载体pPIC9K-PR39-D-E在毕赤酵母中可高效表达抗菌肽PR39,为以后深入研究毕赤酵母高效表达抗菌肽奠定了基础。更多还原

【Abstract】 Objective:To express antibacterial peptide PR39 in Pichia pastoris and determine the activity of product.Methods:Four oligonucleotide fragments were synthesized according to the codon bias of Pichia pastoris,and complete coding sequence was obtained by overlapping PCR,then the sequence was cloned to expression vector pPIC9K.The sequence by Kex2 to the first nucleotide of PR39 was knocked out on the recombinant plasmid pPIC9K-PR39,then the sequence of GGATCCAA in 5’UTR was also deleted by PCR-Restriction Enzyme ligation method,finally a new expression vector named pPIC9K-PR39-D-E was obtained.The vector pPIC9KPR39-D-E was transformed to Pichia pastoris SMD1168,and clones were identified by PCR and than screened with G418 for expression under induction of 0.5%methanol.The expressed product was identified by TricineSDS-PAGE,and determined for antibacterial activity by agarose diffusion test,purified by reversed phase chromatography and ion-exchange column chromatography.Results;PR39 with 4.7kDa and reached 175.6mg/L after purification.It showed antibacterial activity to E.coli DH5α.Conclusion:PR39 was successfully expressed in Pichia pastoris,which laid a foundation of further study on antibacterial peptide.更多还原