巴什拜羊SPLUNC1基因的克隆与真核表达

Cloning and Eukaryotic Expression of SPLUNC1 Gene of Bashibay Sheep

为了克隆巴什拜羊SPLUNC1基因并表达该蛋白,本试验从巴什拜羊肺脏组织中提取总RNA,利用RTPCR方法扩增出SPLUNC1基因开放阅读框序列,将该基因片段插入到真核表达质粒pPIC9K中构建pPIC9K-SPLUNC1重组质粒,然后将pPIC9K-SPLUNC1重组质粒线性化并电击转化入毕赤酵母GS115中,经甲醇诱导蛋白表达后用SDS-PAGE和Western blotting分析鉴定目的蛋白。结果表明,经RT-PCR扩增成功获得大小为748bp的SPLUNC1基因;构建的pPIC9K-SPLUNC1重组质粒经PCR、酶切及测序鉴定与预期结果一致;SDS-PAGE及Western blotting检测鉴定结果表明获得大小为25.53ku的SPLUNC1蛋白。本试验结果为进一步研究巴什拜羊SPLUNC1蛋白的生物学活性奠定基础。

In order to clone Bashibay sheep SPLUNC1 gene and express its protein,total RNA was extracted from lung of Bashibay sheep,RT-PCR was used to amplify SPLUNC1 gene open reading frame sequence,the gene fragment was inserted into eukaryotic expression plasmid pPIC9 Kto construct plasmid pPIC9 K-SPLUNC1,then the plasmid pPIC9 K-SPLUNC1 was linearized and electroporated into Pichia pastoris GS115,the protein was induced by methanol,and the target protein was identified by SDS-PAGE and Western blotting.The results showed that the748 bp SPLUNC1 gene was successfully amplified by RT-PCR;The plasmid pPIC9 K-SPLUNC1 was consistent with expected results after PCR,enzyme digestion and sequencing identification;Detection results of SDS-PAGE and Western blotting showed that the SPLUNC1 protein was successfully expressed with 25.53 ku.This study laid a foundation for further research of SPLUNC1 protein biological activity of Bashibay sheep.