含人极低密度脂蛋白受体基因的重组腺相关病毒的构建
发布时间：2018-09-14 09:39
【摘要】： 目的构建人的极低密度脂蛋白受体(VLDLR)基因,将其包装成含VLDLR基因的重组腺相关病毒(rAAV-VLDLR),并检测其滴度。方法通过设计含有特定酶切位点的引物,从人的骨骼肌中提取RNA,合成cDNA后,扩增人VLDLR全长基因的5’端约1000bp的DNA,并以含人VLDLR基因部分长的质粒为模板,合成人VLDLR全长基因近3’端约1600bp的DNA,通过粘端连接将两片段连接成全长VLDLR,并送基因公司检测其与文献报道的全长人VLDLR相符情况;将构建的人VLDLR基因与重组腺相关病毒的真核表达载体PXXUF1连接,形成人VLDLR的重组腺相关病毒真核表达载体的重组体PXXUF1-VLDLR,鉴定后,将重组腺相关病毒系统的三种质粒Pxx2、Phelper、PXXUF1-GFP(和PXXUF1-VLDLR)大量提取后纯化,以293细胞作为细胞载体,利用磷酸钙共转染法包装rAAV-VLDLR病毒与rAAV-GFP病毒,以rAAV-GFP病毒的包装情况作为rAAV-VLDLR病毒包装效率的参照,用斑点杂交的方法检测两种病毒的滴度。结果1、人极低密度脂蛋白受体基因构建成功,与文献报道基相符;2、PXXUF1-VLDLR构建成功,rAAV-VLDLR病毒与rAAV-GFP病毒的滴度均为4×1011pfu/ml。结论用所构建的人VLDLR基因包装的rAAV-VLDLR病毒达到了要求的滴度,可用于后续糖尿病大鼠和细胞的相关研究。
[Abstract]:Objective To construct human very low density lipoprotein receptor (VLDLR) gene and package it into a recombinant adeno-associated virus (rAAV-VLDLR) containing VLDLR gene and detect its titer.Methods RNA was extracted from human skeletal muscle by designing primers containing specific enzyme digestion sites.After synthesizing the cDNA, the 5'-terminal DNA of the full-length gene of human VLDLR was amplified and the titer of the recombinant adeno-associated virus (rAAV-VLDLR) was determined. The human VLDLR gene was synthesized with a partial length plasmid containing human VLDLR gene as a template. The two fragments were ligated into a full length VLDLR by stick-end ligation and sent to the gene company to detect their conformity with the reported full-length human VLDLR gene. The constructed human VLDLR gene was compared with the recombinant adeno-associated virus eukaryotic expression vector PXXUF1. The recombinant adeno-associated virus eukaryotic expression vector PXXUF1-VLDLR was linked to form human VLDLR. After identification, three recombinant adeno-associated virus system plasmids Pxx2, Phelper, PXXUF1-GFP (and PXXUF1-VLDLR) were extracted and purified. 293 cells were used as cell carriers to package rAAV-VLDLR virus and rAAV-GFP disease by calcium phosphate co-transfection. Results 1. Human VLDLR gene was successfully constructed, which was consistent with the reported base. 2. PXXUF1-VLDLR was successfully constructed, and the titers of rAAV-VLDLR virus and rAAV-GFP virus were 4 *1011 pfu. Conclusion The rAAV-VLDLR virus packaged with the constructed human VLDLR gene has reached the required titer and can be used in the follow-up study of diabetic rats and cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R587.1;R346
本文编号：2242343
[Abstract]:Objective To construct human very low density lipoprotein receptor (VLDLR) gene and package it into a recombinant adeno-associated virus (rAAV-VLDLR) containing VLDLR gene and detect its titer.Methods RNA was extracted from human skeletal muscle by designing primers containing specific enzyme digestion sites.After synthesizing the cDNA, the 5'-terminal DNA of the full-length gene of human VLDLR was amplified and the titer of the recombinant adeno-associated virus (rAAV-VLDLR) was determined. The human VLDLR gene was synthesized with a partial length plasmid containing human VLDLR gene as a template. The two fragments were ligated into a full length VLDLR by stick-end ligation and sent to the gene company to detect their conformity with the reported full-length human VLDLR gene. The constructed human VLDLR gene was compared with the recombinant adeno-associated virus eukaryotic expression vector PXXUF1. The recombinant adeno-associated virus eukaryotic expression vector PXXUF1-VLDLR was linked to form human VLDLR. After identification, three recombinant adeno-associated virus system plasmids Pxx2, Phelper, PXXUF1-GFP (and PXXUF1-VLDLR) were extracted and purified. 293 cells were used as cell carriers to package rAAV-VLDLR virus and rAAV-GFP disease by calcium phosphate co-transfection. Results 1. Human VLDLR gene was successfully constructed, which was consistent with the reported base. 2. PXXUF1-VLDLR was successfully constructed, and the titers of rAAV-VLDLR virus and rAAV-GFP virus were 4 *1011 pfu. Conclusion The rAAV-VLDLR virus packaged with the constructed human VLDLR gene has reached the required titer and can be used in the follow-up study of diabetic rats and cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R587.1;R346
【参考文献】
相关期刊论文 前7条
1 胡舜英;腺相关病毒在基因治疗中的研究进展[J];国外医学.输血及血液学分册;2000年03期
2 屈伸,王剑波,刘志国,邓耀祖,冯宗忱;中国人极低密度脂蛋白受体基因的克隆及其在中国仓鼠卵巢细胞中的表达[J];中国动脉硬化杂志;2000年03期
3 田俊,屈伸,王燕,李映红,王宇哲,宗义强;极低密度脂蛋白受体在泡沫细胞形成中的作用地位[J];中国动脉硬化杂志;2004年05期
4 刘志国,屈伸;极低密度脂蛋白受体研究进展[J];中国生物化学与分子生物学报;2003年06期
5 过健俐,郭伟,姜立,张莉,屈伸;家兔极低密度脂蛋白受体的组织分布研究[J];同济医科大学学报;2001年05期
6 高凌,张木勋,鲍力,屈伸,李春蕊;极低密度脂蛋白受体在糖尿病患者脂肪组织中的分布和表达[J];中华内分泌代谢杂志;2002年03期
7 李桂林,王任直,王欣,窦万臣,张波,田士强,任祖渊,苏长保;质粒辅助重组腺病毒相关病毒的制备和纯化[J];中华医学杂志;2003年14期
，本文编号：2242343
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2242343.html
最近更新
教材专著
