杂合抗菌肽Cecropin P1-Dermaseptin S4在毕赤酵母中的分泌表达及活性鉴定

Secretory expression and biological characterization of Hybrid peptide Cecropin P1-Dermaseptin S4(CP1-DS4)in Pichia pastoris

目的在毕赤酵母中分泌表达具有抗菌活性的杂合抗菌肽Cecropin P1-Dermaseptin S4(CP1-DS4)。方法根据毕赤酵母密码子的偏爱性优化Cecropin P1和Dermaseptin S4两抗菌肽基因序列,通过SOE-PCR拼接,构建克隆载体pMD18T-cp1-ds4并测序;pMD18T-cp1-ds4经EcoRⅠ、XbaⅠ双酶切后构建重组表达载体pPICZαA-cp1-ds4,测序验证开放阅读框(ORF)正确后经SacⅠ线性化处理,然后电转化毕赤酵母X-33(800V,1mm电转化杯,15s电击1次)并利用Zeocin筛选阳性重组子。重组子接种BMGY、BMMY培养基,采用5%的甲醇诱导表达,采用Tricine-SDS-PAGE分析表达上清,表达上清浓缩后进行抑菌试验。结果在本研究中拼接引物经SOE-PCR拼接后得到优化的长度为120bp的Dermaseptin S4基因基因片段,该片段与Cecropin P1基因连接形成约197bp杂合抗菌肽片段,成功构建pPICZαA-cp1-ds4表达载体,电转化获得重组酵母菌pPICZαA-cp1-ds4/X-33,经诱导后可产生分子质量单位约为9.7ku的目的多肽,该多肽对大肠埃希菌和金黄葡萄球菌均有抑制作用。结论杂合肽CP1-DS4可以在毕赤酵母X-33中融合分泌表达,且具有抗菌活性。

Objective Secretory expression of Hybrid peptide Cecropin P1-Dermaseptin S4（CP1-DS4）with antibacterial activity in Pichia pastoris. Methods According to P. pastoris codon bias, the optimized DNA sequences of cecropin Pl and dermaseptin S4 were obtained. Two genes were spliced together by SOE PCR and then constructed and sequenced re- combinant cloning vector pMD18T-cp1-ds4. After pMD18T-cp1-ds4 was digested with EcoR I and Xba I , recombinant fragment was inserted into pPICZαA to construct recombinant expression vector pPICZαA -cp1-ds4. Recombinant expres sion vector pPICZαA-cpl-ds4 was linearized by Sac I after verify the correctness of open reading frame （ORF） by sequen cing and then transformed into P. pastoris X- 33 in lmm cuvette,under the condition of 800V, pulse 15ms one time. Posi- tive recombinants were screened using the Zeocin and induced by BMGY and BMMY medium which contains methanol（0. 5 G v/v）, then to analysis expression supernatant using the Tricine SDS-PAGE, inhibition experiments of concentrated expression supernatant were carried out at last. Results In this study, optimized Dermaseptin S4 gene fragment at long which 120 bp can obtained by SOE PCR splicing splice primers , the fragment can be linked to Cecropin Pl gene fragment to constitute hybrid peptide fragment at long which 197 bp and expression vector pPICZαA-cp1- ds4 was constructed suc- cessfully. Recombinant P. pastoris pPICZαA-cp1-ds4/X-33 was also obtained and has ability to produce interesting pep- tide which molecular weight of approximately 9.7 ku after induced. Concentrated expression supernatant have bactcrio static effect to Escherichla coli and Staphylococcus aureus. Conclusion Low voltage mode can also transforme P. pas toris successfully, hybrid peptide CP1-DS4 can secretory expression in P. pastoris X 33 in a form of fusion protein and have antibacterial activity.