大鼠抗组胺药敏感性细胞色素P450基因的克隆及其反转录病毒载体的构建和鉴定  

HP450 cloning and the construction and identification of its reverse transcriptase recombinant vector

作　　者：陈海英[1] 王乃平[2] 韦锦斌[1] 杨天燕[3] 

机构地区：[1]广西医科大学药理教研室,广西壮族自治区南宁市530021 [2]广西中医学院,广西壮族自治区南宁市530001 [3]广西医科大学第一附属医院药剂科,广西壮族自治区南宁市530021

出　　处：《中国组织工程研究与临床康复》2007年第12期2283-2286,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30360118);广西自然科学基金资助项目(桂科自0640125)~~

摘　　要：目的:构建反转录病毒表达载体pLXSN-HP450。方法:实验于2004-12/2006-05在广西医科大学生化药理实验室完成。提取正常的Wistar大鼠肝组织的总RNA,反转录-聚合酶链反应技术扩增出抗组胺药敏感性细胞色素P450(HP450)基因。并克隆到pMD18-T载体,经蓝白斑筛选后进行聚合酶链反应,酶切,测序鉴定。BamHI,EcoRI双酶切构建好的重组质粒和反转录病毒载体pLXSN在16℃下经T4连接酶连接过夜,并转化到感受态细胞DH5α,以菌液为模板做聚合酶链反应,结合酶切筛选及鉴定阳性重组反转录病毒载体pLXSN-HP450。结果:反转录-聚合酶链反应扩增出HP450基因。重组质粒pMD18-HP450经双酶切后得到1461bp的酶切产物。测序的结果用ClustalW在线与Genebank对比,此目的基因与基因库中的H1基因100%同源。阳性重组载体pLXSN用其菌液聚合酶链反应扩增出目的条带。对重组体pLXSN-HP450进行双酶切得到1461bp和5900bp两条特异性条带。结论:克隆了HP450基因,并成功构建了重组反转录病毒载体pLXSN-HP450,为进一步研究肝癌的基因治疗奠定了基础。AIM： To construct the reverse transcriptase recombinant vector pLXSN-HP450. METHODS： The experiment was conducted in the Biochemical Pharmacology Laboratory, Guangxi Medical University between December 2004 and May 2006. The total RNA was isolated from the normal liver tissue Wistar rat. HP450 gene was amplified by reverse transcriptase-polymerase chain reaction （RT-PCR） and then cloned into pMD18-T vector. The positive clone was obtained by screening following PCR, digestion and sequencing. The recombinant vector digested with restriction enzymes BamHI and EcoRI was ligated with pLXSN at 16 ℃ overnight and transformed to DH5 α. Finally, the positive clone was identified by PCR and enzyme digestion. RESULTS： HP450 gene was cloned by RT-PCR. The product with molecular weight 1 461 bp was obtained after the recombinant vector pMD18-HP450 was digested with restriction enzyme BamHI and EcoRl. The sequencing result compared with Clustal W and Genebank, which showed that this gene shared about 100% nucleotide homology with H1 in the GenBank. The positive recombinant pLXSN was amplified to one gene with molecular weight 1 461 bp. In addition, the products with molecular weight of 1 461 bp and 5 900 bp were obtained after digested with restriction enzyme BamHI and EcoRl. CONCLUSION： The amplification of HP450 gene and construction of recombinant vector pLXSN-HP450 would lay a foundation for further study on gene therapy for liver cancer.

关 键 词：HP450基因 克隆 质粒载体 反转录病毒载体 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]