盘羊杂交羊SP-A基因克隆与真核表达  

Cloning and eukaryotic expression of SP-A in argali hybrid sheep

作　　者：王继雪 孙延鸣[1] Wang Jixue;Sun Yanming(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832003,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003

出　　处：《石河子大学学报（自然科学版）》2018年第3期309-314,共6页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(31460686)

摘　　要：目的为了对盘羊杂交羊肺表面活性物质相关蛋白A(Pulmonary surfactant-associated protein A,SP-A)基因进行克隆和真核表达。方法采集盘羊杂交羊肺组织,提取总RNA,采用RT-PCR的方法克隆SP-A基因的ORF序列,测序正确后将其亚克隆到pPIC9K载体中构建pPIC9K-SP-A真核表达质粒,再将该质粒电转入毕赤酵母GS115中并用甲醇诱导进行真核表达,最后用SDS-PAGE和western blot检测鉴定目的蛋白。结果扩增出的基因片段经测序比对后与SP-A基因的ORF序列一致,构建的真核表达质粒pPIC9K-SP-A经双酶切和测序鉴定成功,真核表达产物用SDS-PAGE分析,可见大小为27.33 k D的目的条带,western blot鉴定也可见1条27.33 kD的目的蛋白条带。结论本研究成功克隆了盘羊杂交羊SP-A基因的ORF序列并对其进行了真核表达,为后续研究SP-A蛋白的结构和功能奠定了基础。Objective In order to clone and express the gene of pulmonary surfactant associated protein A in argali hybrid sheep,total RNA was extracted from the lung tissue of argali hybrid sheep. Methods The ORF sequence of SP-A gene was cloned by RT-PCR and subcloned into pPIC9K vector to construct eukaryotic expression plasmid pPIC9K-SP-A. The plasmid was transfected into Pichia pastoris GS115 and was induced by methanol for eukaryotic expression. Finally, SDS-PAGE and western blot were used to detect and identify the target protein. R esults The results showed that the cloned gene fragment was consistent with the ORF sequence of SP-A gene after sequencing. The eukaryotic expression plasmid pPIC9K-SP-A was identified by double enzyme digestion and sequencing. The eukaryotic expression product was analyzed by SDS-PAGE, and the target band was 27.33 kD. Western blot identification can also be seen a target protein band of 27.33 k D. Conclusion In this study, the ORF sequence of sheep SP-A gene was cloned and expressed in eukaryotic. It lays a foundation for further study on the structure and function of SP-A protein.

关 键 词：盘羊杂交羊 肺表面活性物质相关蛋白A 克隆 真核表达 

分 类 号：S826[农业科学—畜牧学]