摘要
背景:目前大多采用Adeasy系统来构建和包装腺病毒载体,整个过程需要多次酶切,连接和转化筛选,并且在BJ183菌中的重组阳性率很低,还可能存在假阳性的可能,整个过程耗时、繁琐。目的:应用Gateway技术较快速构建大鼠LIM矿化蛋白1基因重组腺病毒载体。方法:从SD大鼠成骨细胞内提取总RNA,并设计LMP-1特异性引物进行RT-PCR,获取LMP-1基因后利用Invitrogen公司的TOPO定向克隆技术创建入门克隆pENTR/D-LMP-1,转化TOP10细菌后挑取阳性克隆摇菌提取质粒,入门克隆再与表达载体pAD/CMV/V5-DEST进行同源重组反应得到病毒载体pAD/CMV/V5-LMP-1,转化细菌后挑选阳性克隆摇菌提取重组病毒质粒,用PacⅠ内切酶线性化后用转染293A细胞后得到重组腺病毒载体。将目的基因与Genebank中LMP-1(基因号:AF095585)一致性BLAST比较,并观察重组腺病毒载体pAd-LMP-1的表达。.结果与结论:总RNA后电泳结果显示所提RNA完整,RNA纯度符合要求。成功获取大鼠LMP-1基因,入门质粒与病毒表达质粒经过酶切鉴定以及测序证明,目的基因与Genebank中LMP-1(基因号:AF095585)作BLAST比较,发现系列一致,没有突变。构建出来的腺病毒载体载体能在293A细胞中包装成功。提示利用Gateway技术构建LMP-1重组腺病毒载体相对简单、快捷地获得pAd-LMP-1。
BACKGROUND：The construction of adenovirus vector using Adeasy system is tedious with poor results due to the multiple restriction enzyme digestion,linkage,and screening.OBJECTIVE：To construct a recombinant adenovirus vector containing LMP-1 gene using Gateway technology.METHODS：Total RNA was extracted from rat osteoblasts and the LMP-1 gene was acquired by RT-PCR,the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology,then the entry clone and the expression vector were used to create the expression clone through the LR recombination reaction;at last the adenovirus expression clone was linearized by PacI and transfected to the 293A cell line.The target gene was compared to LMP-1 in the Genebank（Gene number：AF095585）,and the expression of pAd-LMP-1 was observed.RESULTS AND CONCLUSION：The electrophoresis showed that the extracted RNA was integrated,and the purity was accorded with the requirement.LMP-1 gene was successfully acquired.The entry clone was verified by enzymes digestion,and sequencing.The constructed pAd-LMP-1 recombination adenovirus vector was successfully packaged in 293A cells.This result demonstrated that it is easy and rapid to obtain pAd-LMP-1 by constructing pAd-LMP-1 recombination adenovirus vector using the Gateway technology.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第24期4425-4429,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
深圳市科技计划项目(200602045)~~
