摘要
目的:从鼻咽癌组织中克隆EB病毒潜伏膜蛋白1(LMP1)cDNA,构建真核表达质粒pIRES-LMP1,并检测其在体外的表达。方法:利用分子生物学技术,提取鼻咽癌总RNA,逆转录PCR获得LMP1cDNA,克隆入pDriveCloningVector并测序,利用引物上设计的酶切位点NheI和EcoRI将LMP1插入真核表达质粒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的表达。结果:扩增的LMP1cDNA为1301bp,核酸序列测定证实本实验所构建的质粒正确,该表达质粒在体外转染COS细胞后可表达LMP1分子。结论:实验所构建的pIRES-LMP1表达质粒能在体外表达LMP1分子。
Objective： To clone Epstein- Barr virus latent membrane protein 1 （LMP1） from nasopharyngeal carcinoma（NPC） and construct eukaryon vector pIRES - LMP1 and detect the expression of the plasmid in vitro. Methods： Using cloning technique, total RNA was isolated from human NPC and its cDNA fragments of LMP1 gene was gained by RT- PCR, its cDNA fragments was constructed into the eukaryon vector pIRES. The inserted target gene in the eukaryon vector was verified by nucleotide sequencing. COS cell line was transfected with this eukaryon vector using lipofectin reagent. The expression of LMP1 molecule was detected by RT- PCR and Western blot technique. Results： The eukaryon vector pIRES- LMP1 was obtained by cloning technique. The nucleotide sequences of LMP1 gene in this eukaryon vector had high homology with LMP1 B95 - 8（100% ）. After transfection with this eukaryon vector, the LMP1 molecule was expressed in COS cells. Conclusion： The constructed eukaryon vector oIRES- LMP1 could exoress LMP1 molecule in vitro.
出处
《华西医学》
CAS
2005年第3期426-429,共4页
West China Medical Journal
基金
国家自然科学基金资助项目(30270524)
