摘要
目的从鼻咽癌组织中克隆EB病毒潜伏膜蛋白1(LMP1)与热休克蛋白90β(HSP90β)基因,构建其真核双表达质粒pIRES-LMP1-HSP90β,并检测其在体外的表达。方法提取鼻咽癌组织总RNA,逆转录PCR获得LMP1基因片段和HSP90β基因片段,将二者连接于真核双表达质粒pIRES中,测定序列后,用脂质体包裹转染COS细胞,采用RT-PCR和Westernblot检测LMP1和HSP90β的表达。结果核酸序列测定证实本实验所构建的质粒正确,该双表达质粒在体外转染COS细胞后可表达LMP1和HSP90β分子。结论实验所构建的pIRES-LMP1-HSP90β双表达质粒能在体外同时表达LMP1和HSP90β分子。
Objective This study sought to clone Epstein-Burr virus latent membrane protein 1 （LMP1） and heat shock protein 90 β （HSP90 β） of nasopharyngeal carcinoma （NPC）, to construct the mammalian coexpression plasmid plRES-LMP1-HSP90 β, and to detect the expression of the plasmid in vitro. Methods Total RNA was isolated from human NPC by cloning technique, their eDNA fragments of LMP1 gene and HSP90 gene were gained by RT-PCR, and their eDNA fragments were constructed into the mammalian co-expression plasmid vector pIRES. The inserted target genes in the mammalian co-expression plasmid were verified by nucleotide sequencing. COS cell line was transfected with this mammalian co-expression plasmid using lipofectin reagent. The expression of LMP1 and HSP90 β molecules were detected by RT-PCR and Western blot technique. Results The mammalian expression plasmid plRES-LMP1-HSP90 β was obtained by cloning technique. The nucleotide sequences of LMP1 gene and HSP90 β gene in this mammalian co-expresslon plasmid had high homology with EBV-LMP1 （100%） and human HSP90 β （100%） respectively. After transfection with this mammalian co-expression plasmid, the LMP1 and HSP90 β molecules were expressed in COS cells. Conclusion The constructed mammalian co-expression plasmid plRES-LMP1-HSP90 β can express LMP1 and HSP90 β molecules in vitro at the same time.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第3期339-343,共5页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号30270524)资助
