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[目的]构建人防御素5(HD-5)基因的毕赤酵母表达载体并加以鉴定。[方法]采用PCR技术从人cDNA文库中扩增HD-5基因片段,将HD-5基因扩增产物和毕赤酵母表达载体pPICZαA用EcoRⅠ和XbaⅠ双酶切后连接构成重组质粒,然后转化到E.coliTop10中,筛选阳性克隆,并进行PCR、酶切和序列鉴定。[结果]重组质粒经酶切获得HD-5基因片段大小正确,经序列测定证实HD-5基因正确插入pPICZαA中。[结论]成功构建人防御素5基因的毕赤酵母表达载体。
[Objective] To construct and identify the Pichia pastoris expression vector of human defensin 5 (HD-5) gene. [Method] HD-5 gene fragment was amplified from human cDNA library by PCR. The amplified product of HD-5 gene and pichia yeast expression vector pPICZαA were digested with EcoRⅠ and XbaⅠ to construct a recombinant plasmid and then transformed into Positive clones were screened in E. coli Top10 and subjected to PCR, restriction digest and sequence identification. [Result] The size of HD-5 gene fragments obtained by enzyme digestion was correct. Sequence analysis confirmed that HD-5 gene was correctly inserted into pPICZαA. [Conclusion] Pichia pastoris expression vector with human defensin 5 gene was successfully constructed.