摘要
背景:重组腺病毒质粒的构建方法主要有体外连接法和同源重组法。同源重组法有操作复杂、耗时长、效率低、纯化难的缺点。体外连接法又不可避免非特异性的基因重组及基因突变。目的:应用改良体外连接法构建携带黏结蛋白聚糖1基因的重组腺病毒载体,测定其在心肌成纤维细胞中的感染效率。设计、时间及地点:单一样本实验,于2007-08/2008-02在中山大学附属第二医院林百欣实验中心完成。材料:穿梭载体pShuttle-CMV(含绿色荧光报告基因)和腺病毒骨架质粒pAdxsi购自诺赛基因组研究中心有限公司。方法:核苷酸序列鉴定pCMV-Sport6.1-Sdc1质粒;用KpnⅠ+XhoⅠ从质粒pCMV-Sport6.1-Sdc1切出黏结蛋白聚糖1基因片段,亚克隆至pShuttle-CMV中,形成重组穿梭质粒。用I-CeuI+I-SceI双酶切出重组穿梭质粒中CMV-Sdc1片段,亚克隆至腺病毒基因组质粒中,得到重组腺病毒质粒。将重组腺病毒质粒转染293细胞包装获得重组腺病毒AdCMVSdc1,转化体外培养的心肌成纤维细胞。主要观察指标:用DNA测序、酶切及聚合酶链反应法鉴定重组质粒和病毒,并测定重组腺病毒的滴度和感染效率。结果:①核苷酸序列分析表明,pCMV-Sport6.1-Sdc1质粒正确携带大鼠黏结蛋白聚糖1cDNA;黏结蛋白聚糖1基因被克隆于载体pShuttle-CMV上,以KpnⅠ+XhoⅠ双酶切可回收3kb的克隆片段和5.1kb的载体片段;重组腺病毒质粒用XhoⅠ酶切得到7个片段而空载体仅得到6个片段。②重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用;提取病毒DNA行聚合酶链反应鉴定可扩增出1.13kb的特异性片段;用病毒上清多次重复感染293细胞扩增重组腺病毒后,病毒滴度检测达2.0×1011PFU/mL。③用纯化浓缩后的重组腺病毒以感染复数为100感染心肌成纤维细胞,24h后所有细胞均表达绿色荧光。结论:成功构建了携带大鼠黏结蛋白聚糖1基因的重组腺病毒载体,经纯化浓缩后具有较高的滴度,能有效转染心肌成纤维细胞。
BACKGROUND： In vitro ligation and homologous recombination are two primary methods to Construct recombinant adenovirus vector. The homologous recombination is complex, time-cost, low efficient and difficulty to purify. The in vitro ligation leads to nonspecific gene recombination and gene mutation. OBJECTIVE： To construct rat Sdc 1 gene recombinant adenovirus vector using modified in vitro ligation and evaluate its infection efficiency. DESIGN, TIME AND SETTING： Single sample experiment was performed at the Lin Bai-xin Experimental Center, Second Hospital of Sun Yat-sen University from August 2007 to February 2008. MATERIALS： Shuttle plasmid pShuttle-CMV containing green fluorescent protein gene and adenoviral backbone plasmid pAdxsi were bought from SinoGenoMax Company Limited. METHODS： pCMV-Sport6.1-Sdc 1 plasmid was detected by sequencing. Sdc I eDNA segment was liberated from the cloning vector of pCMV-Sport6.1-Sdcl via Kpn I ＋ Xbo I, and subcloned into pShuttle-CMV, which was digested by I-CeuI ＋ I-SceI double enzyme and subcloned into adenoviral plasmid to form recombinant adenovirus plasmid. Recombinant adenovirus plasmid was transfected into 293 cell lines by liposome, and the recombinant adenovirus AdCMVSdc 1 was packaged and transfected into cardiac fibroblasts. MAIN OUTCOME MEASURES： Sequencing, enzyme digestion and PCR were used to detect recombinant plasmid and adenovirus and the titer and infection efficiency of AdCMVSdc 1 was evaluated. RESULTS： Sequencing results indicated that pCMV-Sport6.1-Sdc 1 plasmid contained rat Sdc 1 eDNA correctly. Cloned sequence about 3 kb was obtained by Kpn I ＋ Xho I digestion after Sdcl eDNA segment was cloned into pShuttle-CMV. Recombinant adenovirus plasmid was cut into seven fragments while empty vector gained only six fragments after digested by Xho I. The recombinant adenovirus was pathogenic to 293 cells after recombinant adenovirus plasmid was packaged in it. Sdcl cDNA （1.13 kb） was amplified by PCR with virus titer of 2.0×10^11 PFU/ml after transfected 293 ceils with superuatant. The efficiency of recombinant adenovirus infecting cardiac fibroblasts was 100% when multiplicity of infection was 100. CONCLUSION： The adenovirus vector with rat Sdcl eDNA was established successfully, and high titer was gained after purification and concentration. The vector can effectively transfect cardiac fibroblasts.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第42期8290-8293,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
作者简介
雷娟 ，女，1977年生，广东省乐昌市人，汉族，中山大学在读博士，主治医师，主要从事冠心病的基础与临床研究。Leijuan_1@hotmail.com
通讯作者：伍卫，教授，博士生导师，中山大学附属第二医院心血管内科，广东省广州市510120 wuwei@medmail.com
