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| 靶向克隆法构建pET15b-VP3原核表达载体 | |
| 引用本文： | 邓守恒,柯贤柱,石小燕,蔡召忠,杨敬宁,黄永章,陈萍. 靶向克隆法构建pET15b-VP3原核表达载体[J]. 成都医学院学报, 2012, 7(3): 393-395 |
| 作者姓名： | 邓守恒 柯贤柱 石小燕 蔡召忠 杨敬宁 黄永章 陈萍 |
| 作者单位： | 1. 湖北医药学院附属人民医院肿瘤中心,十堰,442000 2. 湖北医药学院附属人民医院医务处,十堰,442000 3. 武汉市中心医院实验中心,武汉,430014 4. 湖北医药学院免疫教研室,十堰,442000 5. 湖北医药学院附属人民医院临床医学研究所,十堰,442000 |
| 基金项目： | 湖北省卫生厅基金，湖北医药学院创新基金，十堰市科技局基金 |
| 摘 要： | 目的探索利用PCR产物靶向克隆法构建能够表达凋亡素的pET15b-VP3原核表达载体。方法以PGEX-6P-1/TAT-VP3质粒为模板,PCR扩增VP3基因的366bp片段,应用靶向克隆酶,将VP3基因插入到线性pET15b,得到重组表达载体pET15b-VP3,经过筛选,挑选出阳性克隆进行XhoⅠ和BamHⅠ双酶切,图谱分析和重组质粒核苷酸序列分析以鉴定所构建的原核表达载体。结果 PCR产物经过电泳鉴定可见366bp特征性的VP3片段,重组质粒经双酶切电泳获得了一个大小为366bp的VP3片段,重组质粒测序证实VP3cDNA编码区成功地插入了pET15b,且其序列与GeneBank中VP3cDNA序列完全一致。结论靶向克隆法可快速,高效地构建pET15b-VP3原核表达载体,为进一步研究VP3药用价值奠定了基础。 |
| 关 键 词： | pET15b-VP3 靶向克隆 原核表达 |
Construction of pET15b-VP3 Gene Prokaryotic Expression Vector by LP Recco PCR Cloning |
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| DENG Shou-heng , Ke Xian-zhu , SHI Xiao-yan , CAI Zhao-zhong , YANG Jing-ning , HUANG Yong-zhang , CHEN Ping. Construction of pET15b-VP3 Gene Prokaryotic Expression Vector by LP Recco PCR Cloning[J]. Journal of Chengdu Medical College, 2012, 7(3): 393-395 | |
| Authors: | DENG Shou-heng Ke Xian-zhu SHI Xiao-yan CAI Zhao-zhong YANG Jing-ning HUANG Yong-zhang CHEN Ping |
| Affiliation: | 1. Center of oncology , RenMing Hospital, Hubei University of Medicine, Shi yan , Hubei 442000, China ; 2. Medical office RenMing Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China 3. Experiment Center, Wu Han Central Hospital, WuHan , Hubei 430014, China 4. Department of Immunology, Hubei University of Medicine, Shiyan, Hubei 442000, China ; 5. Experiment Center, RenMing Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China) |
| Abstract: | Objective To construct the prokaryotic expression vector of pET15b-VP3 by the method of LP Recco PCR Cloning. Methods The plasmid PGEX-6P-1/TAT-VP3 was the template,the primer containing special enzyme was designed, VP3 fragment was amplified by PCR, and then was cloned into Linearized pETlSb by LP Recco PCR Cloning. The recombinants plasmid pET15b-VP3 were identified by restriction endonucleases digest analysis and sequence analysis. Results The sequence of VP3 segment was amplified and identical with that published in GenBank. The target gene VP3 was successfully cloned into Linearized pET15b. Conclusion LP Recco PCR Cloning can efficiently and conveniently construct high quality prokaryotic expression vector of pET15b-VP3 gene, which has laid the foundation for further researches on the medical values of VP3 protein. |
| Keywords: | pET15 b-VP3 Gene LP Recco PCR cloning Prokaryotic expression |
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