携带人降钙素基因相关肽基因的重组逆转录病毒的构建及鉴定  

Construction and identification of recombinant retroviral vector pLNCX2-human caicitonin gene-re- lated peptide a

作　　者：王义生[1] 皮国富[1] 王亚寒[1] 赵国强[2] 李月白[3] 

机构地区：[1]郑州大学第一附属医院骨科河南省高等学校临床医学重点学科开放实验室,450052 [2]郑州大学基础医学院微生物与免疫学教研室 [3]生物化学与分子生物学教研室

出　　处：《中华实验外科杂志》2012年第5期940-942,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81171776）

摘　　要：目的构建、鉴定携带人降钙素基因相关肽a（hCGRPa）的重组逆转录病毒载体pLNCX2-hCGRPa。方法采用基因工程技术，双酶切消化后回收438bp的hCGRPacDNA片段和6．1kb的pLNCX2载体片段，纯化后按摩尔比7：1混合，将降钙素基因相关肽基因片段克隆至逆转录病毒载体pLNCX2上，序列测定、对比。鉴定后用脂质体法转染PT67细胞进行病毒包装扩增，感染NIH3T3细胞，观察细胞克隆，测定病毒滴度。结果酶切、测序结果与hCGRPa基因重组逆转录病毒载体的预期结果一致，分别为6．1kb和438bp，与空载体和目的基因大小相符。包装、扩增获得病毒滴度达1．7×10^6pfu／ml，对NIH3T3细胞有较高的感染效率。结论成功构建重组逆转录病毒pLNCX2-hCGRPoL，为进一步的基因治疗研究奠定了实验基础。Objective To construct and identify the recombinant retroviral vector encoding human calcitonin gene-related peptidect （hCGRPa） gene. Methods Using genetic engineering techniques, the fragment of hCGRPct eDNA and the fragment of pLNCX2 carrier, respectively 438 bp and 6. 1 kb, were recovered after double digestion, then mixed according to molar ratio of 7：1 after purification. The hCGRPct gene was cloned into the retroviral vector pLNCX2. High titer viral particles of pLNCX2- hCGRPa were obtained after transfection of PT67 cells by Lipofectine 2000. Then the recombinant retrovirus was transfeeted into NIH3T3 cells and the titer was measured. Results Restriction, and sequencing analysis confirmed that the hCGRPa eDNA was successfully inserted into the retroviral vector. The titer of recombinant retro- virus with hCGRPa gene was 1.7 × 10^6 pfu/ml and the retrovirus was effectively transfected into NIH3T3 cells. Conclusion The recombinant retroviral vector pLNCX2-hCGRPa had been constructed successfully, which can be used for further investigation on gene therapy.

关 键 词：逆转录病毒 降钙素基因相关肽 基因克隆 股骨头坏死 

分 类 号：R6[医药卫生—外科学]