摘要
目的构建含有人Toll样受体2(TLR2)胞外区基因的重组腺病毒载体并进行鉴定。方法应用RT-PCR方法从人外周血单个核细胞(PBMC)中扩增TLR2胞外区全长基因,克隆入pMD18-T载体并经测序验证后,通过KpnⅠ和HindⅢ双酶切将目的片段定向克隆至经相同处理的pAdTrack-CMV腺病毒穿梭质粒中。将构建正确的重组质粒pAdTrack-CMV-TLR2用PmeⅠ酶切线性化后转化感受态AdEasier-1细菌,在大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy-1进行细菌内同源重组,获得重组腺病毒质粒。将鉴定正确的重组质粒pAd-TLR2经PacⅠ酶切线性化后,以脂质体法转染至293细胞中进行包装并扩增病毒。观察绿色荧光蛋白(GFP)的表达并测定病毒滴度,用PCR法鉴定重组腺病毒。结果RT-PCR扩增得到的目的基因片段与GenBank中的序列一致,经酶切鉴定及PCR检测证实携带人TLR2胞外区全长基因的重组腺病毒制备成功,获得高滴度(3×109pfu/ml)的重组腺病毒。结论成功制备了表达人TLR2胞外区基因的重组腺病毒,为后续研究奠定了基础。
Objective To construct and identify the recombinant adenovirus vector containing the Toll-like receptor 2 （TLR2） extracellular domain gene of human. Methods Human TLR2 extracellular domain eDNA was amplified by RT PCR from peripheral blood mononuelear cells （PBMCs） and inserted into pMD18-T vector. After being confirmed by enzyme digestion and sequencing, the DNA fragment, digested with Kpn Ⅰ and Hind Ⅲ, was directionally cloned into adenovirus shuttle plasmid pAdTrack-CMV. After linearization by PmeⅠ digestion, the recombinant plasmid pAdTrack CMV-TLR2 was transformed into competent AdEasier-1 germs and then bomologically recombined with an adenoviral backbone plasmid pAdEasy 1 in bacteria BJ5183 to obtain the recombinant adenovirus plasmid. After confirmation, the recombinant adenovirus plasmid pAd TLR2 was linearized with Pac I digestion and transfected into 293 cells via liposome, and then package and adenovims amplification were performed. The expression of green fluorescent protein （GFP） was observed, the virus titer was determined and the recombinant adenovirus was identified by PCR. Results The gene fragment obtained by RT-PCR was of the same sequence as in GenBank. It was certified by restricted endonuclease digestion and PCR analysis that the recombinant adenovirus conraining the TLR2 extracellular domain gene of human had been successfully constructed with a satisfactory high titer of 3 × 10^9pfu/ml. Conclusion The recombinant adenovirus containing TLR2 extracellular domain gene of human has been successfully constructed, which lays a foundation for further study on the structure and biological activity of TLR2.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第4期470-473,共4页
Medical Journal of Chinese People's Liberation Army
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”开放基金资助项目(2006B-2)
关键词
TOLL样受体2
腺病毒科
同源重组
Toll like receptor 2
adenoviridae
homologous recombination
