TLR4全长及其截断体重组腺病毒的制备和功能鉴定

制备脂多糖 (LPS)Toll样受体 4 (TLR4 )全长及其胞内段缺失的TLR4截断体 (ΔTLR4 )的绿色荧光蛋白重组腺病毒并鉴定其功能 .用PCR方法扩增TLR4及ΔTLR4基因片段 ,酶切后亚克隆至腺病毒穿梭质粒中 ,形成带有目的基因的穿梭载体pAdTrack TLR4和pAdTrack ΔTLR4 .用BJ5 1 83细菌同源重组法将目的基因重组于腺病毒骨架载体中 ;将重组腺病毒质粒用PacⅠ酶切线性化后 ,用脂质体法转染HEK 2 93细胞进行腺病毒的包装扩增 .将重组腺病毒感染CHO K1细胞 ,采用荧光毒酶报告基因方法检测其对LPS诱导NF κB激活的影响 .酶切及测序表明 ,TLR4全长及其截断体ΔTLR4的重组腺病毒载体构建正确 .荧光素酶报告基因检测结果表明 ,TLR4全长及其截断体的重组腺病毒感染细胞对LPS诱导的反应具有不同的影响 ,Ad ΔTLR4明显抑制了LPS引起的NF κB激活 (P <0 0 5 ) ,Ad TLR4则使LPS引起的NF κB活性增强 (P <0 0 5 ) .LPS对细胞的激活作用依赖于TLR4的结构完整性

Preparation and Functional Identification of Recombinant Adenovirus of Full-length and Trancated Form of Toll-like Receptor 4

The recombinant adenoviruses that expresses the full length and trancated form of toll like receptor 4 (TLR4 and ΔTLR4) were constructed for the identification of their function. The sequences of TLR4 and Δ TLR4 were amplified by PCR from a TLR4 containing plasmid pcDNA3 TLR4 followed by subcloning of the fragments into an adenovirus shuttle vector pAdTrack to form transfer plasmids, pAdTrack TLR4 and pAdTrack Δ TLR4. After linearization with Pme I, pAdTrack TLR4 and pAdTrack Δ TLR 4 were cotransformed into BJ5183 bacteria that were pretransformed with adenovirus genomic plasmid of pAdEasy 1. The positive recombinant adenovirus plasmids were digested with Pac I and transfected into HEK 293 cells for the packaging of recombinant adenovirus particles. After infection of the resultant viruses in CHO K1 cells, LPS induced NF κB trans activity was examined by a routine Luciferase report gene test. The recombinant adenoviral vectors were identified containing a full length or trancated fragments of TLR4. From the experiments of the report gene, it was shown that TLR4 recombinant adenovirus has enhanced the activation of NF κB that was induced by LPS in CHO K1 cells ( P <0 05), while ΔTLR4 recombinant adenovirus inhibited the activation of NF κB that was induced by LPS in CHO K1 cells significantly ( P <0.05). The full length and trancated form of TLR4 recombinant adenoviruses were successfully constructed. The infection of these recombinant adenoviruses in cells showed different effects on LPS induced cellular response. LPS induced cellular activity depends on the integration of whole structure of TLR4.