人防御素-5基因的克隆和真核表达载体的构建

目的对人防御素5(HD-5)基因进行克隆,构建真核表达载体HD5-pPICZαA。方法从人cDNA文库中PCR扩增HD-5基因片段,用EcoRⅠ和XbaⅠ分别双酶切HD-5基因扩增产物和毕赤酵母表达载体pPICZαA,用T4DNA连接酶连接目的基因片段和pPICZαA,然后转化到E.coli Top10中,Zeocin筛选转化子并进行PCR、酶切和序列鉴定。结果提取阳性克隆的重组质粒,EcoR Ⅰ和Xba Ⅰ双酶切后跑电泳获得预期条带;同时重组质粒经序列测定,证实HD-5基因正确插入pPICZαA中,插入位置、方向均正确。结论成功构建人防御素5基因的真核表达载体HD5-pPICZαA,为HD-5蛋白的真核真核表达奠定基础。

Cloning of human defensin-5 gene and construction of eukaryotic expression vector

Objective To construct human defensin-5（HD-5） gene into eukaryotic expression vector HD5-pPICZαA. Methods HD-5 was amplified from human cDNA by PCR. HD-5 gene product fragments and Pichia pastoris expression vector pPICZαA were double-digested by EcoRⅠand XbaⅠand connected by T4 DNA ligase. Ligation mixtures were transformed into competent E.coli Top10, and positive clones were screened with Zeccin. Transformants were identified by enzyme digesting, PCR and sequencing analysis. Results It was proved that HD-5 gene segment was inserted into pPICZαA correctly after identification with PCR enzyme cutting and sequencing analysis. Conclusion Eukaryotic expression vector HD5-pPICZαA was successfully constructed, which would provide a exprimental basis for further studyon the HD-5 expression in Pichia pastoris.