重组人神经生长因子β腺病毒的构建及鉴定  

Construction and Identification of the Recombinant Adenovirus AdhNGF-β

作　　者：冯波[1] 李青旺[1] 肖波[1] 韩增胜[2] 支旭勃[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]燕山大学环化学院生物工程系,河北秦皇岛066004

出　　处：《安徽农业科学》2008年第2期457-459,511,共4页Journal of Anhui Agricultural Sciences

基　　金：河北省秦皇岛市科技局转基因动物生产基因工程药物研究项目(DO8)

摘　　要：[目的]克隆带有His标签的人神经生长因子β基因(human nerve growth factor beta,hNGF"β),并构建hNGF"β与EGFP基因复制缺陷型腺病毒载体,为hNGF蛋白的表达、纯化及对其进行神经损伤的应用研究奠定基础。[方法]用Trizol试剂提取人胎肝总RNA,应用RT-PCR方法以自行设计的带有His标签的引物来扩增NGF"β基因;将所扩增的NGF"β基因经酶切、纯化,与IRES-EGFP片段共同插入到Pshuttle-cmv载体中,测序后,利用基因同源重组技术构建hNGF基因的复制缺陷型腺病毒载体AdNGF"β,经鉴定,转染HEK293细胞,观察EGFP表达并检测重组腺病毒的病毒滴度。[结果]克隆的hNGF基因序列与GenBank中的hNGF基因序列完全一致并在3′端携带上His标签;重组腺病毒载体AdNGF-β经PacⅠ酶切得到大于23.0和4.5或2.9kb大小的2个片段,表明同源重组成功。2种重组腺病毒经脂质体介导转染293细胞后,均可观察到绿色荧光蛋白。收获病毒并测定感染滴度分别为1.00×109和0.99×109pfu/ml。[结论]克隆了带有His标签的hNGF"β基因,并成功构建了重组腺病毒载体,为hNGF蛋白的表达、纯化及对其进行神经损伤研究奠定基础。[Objective] The aiming was to clone human nerve growth factor-β （hNGF-β） gene with His-tag and construct the recombinant adenovirus plasmid containing hNGF-β and EGFP gene for further studying expression, purification of hNGF -β and nerve injuries treated by hNGF-β. [Method] Total RNA was extracted by Trizol from human embryonic liver, the hNGF-β gene was amplified by RT-PCR using the primers contained His-tag based on the published sequence of hNGF-β. The double-digested PCR product was purified and sequenced, the recombinant adenovirus vector Ad-hNGF-β-EGFP was constructed with hNGF-β and EGFP by molecular cloning technique and identified. The vector was transfected into HEK293 cell and then viral title was checked by EGFP. [Result] Cloning hNGF-β gene sequence was the same as that of published sequence. The analysis of recombinant adenovirus plasmid digested by restrictive endonuclease Pac Ⅰ confirmed that it was constructed successfully. There were 2 kinds of correct recombinant adenovirus plasmid which could be cut out 23.0 and 2.9 or 4.5 kb. The large fragments of 23.0 kb were transduced into HEK293, respectively. Twenty-four hours after transduction, the fluorescence was observed in 293 cells. The viral rites were 1.00×10^9 and 0.99×10^9 pfu/ml, respectively, which were checked by EGFP. [Conclusion] The hNGF-β gene was cloned from liver and a recombinant adenovirus vector containing human hNGF-β and EGFP gene was constructed successfully. It laid the foundation for further study of expression and purification of hNGF-β as well as nerve injuries treated by hNGF-β.

关 键 词：人神经生长因子β 基因克隆 腺病毒 

分 类 号：Q78[生物学—分子生物学]