克隆人血小板源性生长因子B基因构建腺相关病毒载体及其病毒滴度测定

Construction and titer assay of adeno-associated virus vector by cloning the human platelet-derived growth factor B gene

目的:克隆人血小板源性生长因子B基因于腺相关病毒载体中,以获得高感染滴度的重组人血小板源性生长因子B基因腺相关病毒。方法:实验于2005-10/2006-11在青岛大学医学院附属医院中心实验室完成。①健康足月产妇胎盘组织由青岛大学医学院附属医院妇产科提供,产妇及其家属均知情同意。DH5α宿主菌由青岛大学医学院附属医院中心实验室制备保存。②Trizol试剂提取健康足月产妇胎盘组织总RNA,反转录-聚合酶链反应一步法克隆人血小板源性生长因子B基因全长开放读框。聚合酶链反应产物定向克隆于腺相关病毒载体,重组人血小板源性生长因子B基因腺相关病毒载体进行双酶切和测序鉴定。③采用磷酸钙转染法将重组人血小板源性生长因子B基因腺相关病毒载体、腺相关病毒包装载体,腺相关病毒辅助载体共转染腺相关病毒293细胞,包装得到重组腺相关病毒并回收病毒原液,显微镜下观察细胞及培养基的变化。④以携带β-半乳糖苷酶基因的腺相关病毒作为报告基因同重组人血小板源性生长因子B基因腺相关病毒载体一样共转染相关病毒293细胞,对报告基因行X-Gal染色,通过蓝染的细胞计算病毒滴度。结果:①重组人血小板源性生长因子B基因腺相关病毒载体酶切鉴定及序列分析:聚合酶链反应产物经电泳证明大小正确,重组人血小板源性生长因子B基因腺相关病毒载体酶经双酶切和测序鉴定证实将血小板源性生长因子B基因正确插入。②腺相关病毒293细胞转染过程中病毒颗粒的产生:腺相关病毒293细胞转染6h后培养基底部出现无数小黑色颗粒,为加入的转染复合物颗粒。24h后腺相关病毒293细胞部分变圆,随着病毒增殖,细胞成串浮起,出现细胞病理效应。48h后细胞变形,逐渐从壁上脱落。72h后培养基的颜色从红色变化为橙色或黄色。③病毒滴度检测:通过携带β-半乳糖苷酶基因的腺相关病毒感染细胞后X-gal染色计数,测定重组腺相关病毒滴度为1×1010 L-1。结论:成功构建表达人血小板源性生长因子B基因的重组腺相关病毒载体,为进一步研究血小板源性生长因子B基因治疗角膜病和相关细胞系与组织的增殖提供实验基础。

AIM： To clone human platelet-derived growth factor B gene into adeno-associated virus vector （AAV） and harvest high titer recombinant adeno-associated virus for infecting corneal endothelial cell. METHODS： The experiment was carded out at the Laboratory of Molecular Biology, Affiliated Hospital of Qingdao University Medical College from October 2005 to November 2006. ①Healthy mature placenta was provided by the Department of Gynaecology and Obstetrics, Affiliated Hospital of Qingdao University Medical College, and the informed consent was obtained; DH 5α was offered by Laboratory of Molecular Biology of Affiliated Hospital of Qingdao University Medical College. ②The total RNA was extracted from healthy mature placenta by Trizol. The full-length open reading frame of human platelet-derived growth factor B gene was amplified by RT-PCR. The PCR products were inserted into virus vector, and the recombinant adeno-associated virus vector was identified by double enzyme digestion and DNA sequencing.③The packaging of recombinant adeno-associated virus vector with human platelet-derived growth factor B gene was based on the pAAV-RC and pHelper co-transfection into AAV-293 cells by calcium phosphate-based protocol, and the stock solution of virus was retrieved. Meanwhile, monitoring the phenotypic changes in the cells and culture medium were observed by microscope. ④Like recombinant adeno-associated virus vector with human platelet-derived growth factor B gene, the adeno-associated virus vector with LacZ gene was transfected jointly into AAV-293 cells, too. The titer of recombinant adeno-associated virus vector with LacZ gene was calculated by means of cells X-Gal staining that expressed the report gene LacZ. RESULTS： ①The results of double enzyme digestion and DNA sequencing of recombinant adeno-associated virus vector with human platelet-derived growth factor B gene showed that the full-length open reading frame of human platelet-derived growth factor B gene had been inserted into adeno-associated virus vector correctly. ②After 6 hours transfection of AAV-293 cells, there were countless blank particles that was the transfecting complex in the bottom of culture medium. Twenty-four hours later, part of AAV-293 cells turned into round with the proliferation of virus, the cells floated. Forty-eight hours later, cells degraded, and gradually detached from the plate. Seventy-two hours later, the color of the medium changed from red to orange or yellow compared with negative control. ③The titer of the recombinant viral particles with human platelet-derived growth factor B gene was about 1×10^10 particles/L by calculating the cells stained with X-Gal. CONCLUSION： Recombinant adeno-associated virus vector with human platelet-derived growth factor B gene is constructed correctly, which would benefit to further study on its proliferation effect on mammalian corneal endothelial cells or related cell line and tissues.