【摘要】 目的克隆人热休克蛋白90β(HSP90β)基因,构建其真核表达载体pcDNA3.1(+)/hHSP90β,并检测其在体外的表达情况,为下一步研究鼻咽癌中HSP90β功能奠定基础。方法提取鼻咽癌总RNA,并经RT-PCR获得hHSP90βcDNA。用纯化的hHSP90βcDNA与PGEM Easy T Vector连接,构建中间载体PGEM-hHSP90β。将PGEM-hHSP90β及pcDNA3.1(+)质粒经AflII和Xbal双酶切、胶回收纯化酶切片段后,体外连接酶切片段,构建hHSP90β真核表达载体pcDNA3.1(+)/hHSP90β。经酶切和测序后,用脂质体包裹转染COS细胞,采用RT-PCR和Western blot检测HSP90β的表达。结果酶切及测序鉴定表明,hHSP90β与pcDNA3.1(+)体外重组成功,命名为pcDNA3.1(+)/hHSP90β。该表达质粒在体外转染COS细胞后可表达HSP90β分子。结论采用体外重组技术,成功地将人HSP90β插到了真核表达载体pcDNA3.1(+)中;pcDNA3.1(+)/hHSP90β表达质粒能在体外表达HSP90β分子。更多还原

【Abstract】 Objective To establish a construct that can express human HSP90β in eukaryote cells in vitro.Methods Total RNA was isolated from human NPC and HSP90βcDNA was prepared by RT-PCR.The purified HSP90β fragment was cloned into PGEMT Easy T Vector,and then sub-cloned into pcDNA3.1(+) vector.The resulted pcDNA3.1(+)/hHSP90β plasmid was verified by restriction enzyme digestion and DNA sequencing.COS cells were transfected with this plasmid and the expression of HSP90β was examined by RT-PCR and Western blot analysis.Results Sequencing and restriction enzyme digestion analysis showed HSP90β cDNA was correctly cloned into vector pcDNA3.1(+).HSP90β was expressed in COS cells transfected with pcDNA3.1(+)/hHSP90β plasmid.Conclusion The constructed eukaryote expression plasmid pcDNA3.1(+)/hHSP90β could express HSP90β in eukaryote cells in vitro.更多还原