HPC2突变体真核表达载体的构建

摘要

目的 构建在哺乳动物细胞表达的HPC2真核表达突变载体,并在U2OS细胞中表达.方法 从人U2OS细胞中克隆HPC2cDNA片段,并利用Gateway技术构建pcDNA3.2/V5/HPC2质粒.将质粒用脂质体瞬时转染U2OS细胞,细胞裂解后用Western blot分析HPC2的表达情况.设计突变引物,以HPC2质粒为模板进行PCR,将PCR产物连接并转化扩增,得到特定位点突变的pcDNA HPC2质粒.结果 PCR、酶切、测序均表明pcDNA-V5/HPC2突变质粒构建成功,并可在U2OS细胞中表达.结论 成功构建了pcDNA-V5/HPC2突变真核表达载体,为研究HPC2的功能提供了有力的工具.%Objective To construct mutated HPC2 eukaryotic expression vector and to express it in U2OS cells. Methods By means of Invitrogen Gateway system, wild type HPC2 cDNA was obtained from U2OS cells mRJMA by RT-PCR, and constructed into pcDNA 3.2/V5 vector. The expression of HPC2 was confirmed by western blot. Mutated HPC2 were made according to phosphorylau'on site prediction software analysis (NetPhosKl .0) and constructed with a mutation kit (TaKaRa MutanBEST). Results Double restriction enzyme digestion and DNA sequencing testified the correct sequence and direction of mutated HPC2/V5 eukaryotic expression vector. Conclusion The successfully constructed mutated HPC2/V5 eukaryotic expression vector could provide a beneficial tool for the research of HPC2/V5.