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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 region gene recombinant adenovirus vector. Methods The pUCm-TLR4 plasmid was used as a template to amplify the extracellular domain of TLR4 by polymerase chain reaction (PCR). The fragment was recovered and ligated into the shuttle plasmid pAdTrack-CMV to obtain the recombinant adenovirus plasmid pAdTrack-CMV-TLR4. Hind Ⅲ double enzyme digestion, sequencing identification, the identification of the correct plasmid was digested with PmeI linearized, transformed into adenovirus backbone plasmid pAdEasy-1 competent bacteria BJ5183 for homologous recombination, positive Kanam resistance screening The plasmids were cloned and transfected into 293 cells by liposome. The recombinant adenovirus was identified by observing the expression of green fluorescent protein (GFP), PCR technology and Western blot, and the virus titer was determined. Results Human TLR4 extracellular domain gene recombinant adenovirus vector was constructed correctly with a titer of 3.2 × 109pfu / mL. Conclusion The human recombinant adenovirus vector of TLR4 extracellular region was successfully constructed, which laid the foundation for further experiments.