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1人TLR4胞外区基因重组腺病毒载体的构建与鉴定 被引量:3

Construction and identification of recombinant adenovirus vector containing human TLR4 extracellular domain gene
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摘要 目的构建并鉴定人TLR4胞外区基因重组腺病毒载体。方法以pUCm-TLR4质粒为模板,运用PCR技术扩增TLR4胞外区目的基因片段,片段回收后经酶切连接穿梭质粒pAdTrack-CMV上,获得重组腺病毒质粒pAdTrack-CMV-TLR4.通过KpnⅠ和HindⅢ双酶切,测序鉴定后,将鉴定正确的质粒经PmeI酶切线性化后,转化到含有腺病毒骨架质粒pAdEasy-1的感受态细菌BJ5183中,进行同源重组,卡那抗性筛选阳性克隆,提取质粒,用脂质体介导转染293细胞,通过观察绿色荧光蛋白(GFP)的表达及PCR技术,Western blot等方法鉴定重组腺病毒,并进行病毒滴度测定。结果人TLR4胞外区基因重组腺病毒载体构建正确,病毒滴度为3.2×109pfu/mL。结论成功构建了人TLR4胞外区基因重组腺病毒载体,为下一步实验奠定基础。 Objective To construct and identify human TLR4 extracellular recombinant adenovirus.Methods TLR4 extracellular domain gene was amplified by PCR with pUCm-TLR4 plasmid as template,and then connected to the shuttle vector pAdTrack-CMV for constructing recombinant adenovirus shuttle plasmid pAdTrack-CMV-TLR4 by enzyme digestion.After sequencing,the plasma was linearized with pmeI and cotransformed into E.coli.BJ5183 cells containing pAdEasy-1 plasmid to undergo homologous recombination.After been screened by s...
出处 《免疫学杂志》 CAS CSCD 北大核心 2009年第4期457-460,共4页 Immunological Journal
基金 全军"十一五"面上项目(2006B060) "创伤 烧伤与复合伤国家重点实验室"开放基金(2006B-2)资助
关键词 TLR4基因 腺病毒载体 胞外区 TLR4 adenovirus vector extracellular domain
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