中国组织工程研究与临床康复2011，Vol.15Issue(20)：3693-3696,4.DOI:10.3969/j.issn.1673-8225.2011.20.020
沉默Toll样受体4基因表达的RNAi慢病毒载体的构建
Construction of a lentiviral vector for RNA interference targeting human toll-like receptor 4 gene
摘要
Abstract
BACKGROUND: As the most significant receptor mediating endotoxin or lipopolysaccharide response, toll-like receptor 4 (TLR4) plays a key role in the signaling pathway of inflammation induced by endotoxin.OBJECTIVE: To construct a lentiviral RNAi vector that is capable of knocking down TLR4 gene in human umbilical vein endothelial cells (HUVECs).METHODS: Short hairpin RNA (shRNA) sequence targeting Human TLR4 gene was designed using the Invitrogen online designing software. After synthesis and annealing, the double-stranded oligo nucleotides (ds oligo) were cloned into pENTRTM/H1/TO vectors and the resulting entry clones were identified by sequencing. Then, a recombinant reaction was performed to transfer the RNAi cassette from the entry plasmids into pLenti4/BLOCK-iTTM-DEST vectors to create the expression clone. Recombinant lentivirus was harvested from 293FT cells contransfected with the expression plasmids and the ViraPowerTM packing Mix. HUVEC cells were infected with the recombinant lentivirus and TLR4 expression in these cells was subsequently detected by western blot.RESULTS AND CONCLUSION: Recombinant lentivirus expressing shRNA targeting the TLR4 gene was successfully constructed with the titer of 8.7×106 U/ml. Western blot result showed that the expression of TLR4 in lentivirus-infected HUVEC cells was significantly lower than that in the control cells. The lentiviral RNAi vector with the capability of knocking down TLR4 gene in HUVEC cells has been successfully constructed, providing a basis for further study on the relationship between inflammation and TLR4 gene.关键词
Toll样受体4/RNA干扰/慢病毒/炎症/组织构建分类
医药卫生引用本文复制引用
阮静,王旭,李煜生,刘爱华,姜勇..沉默Toll样受体4基因表达的RNAi慢病毒载体的构建[J].中国组织工程研究与临床康复,2011,15(20):3693-3696,4.基金项目
课题受国家自然科学基金重点项目(NO.81030055)、国家自然科学基金委员会-广东省人民政府自然科学联合基金(NO.U0632004)、长江学者和创新团队发展计划(NO.IRT0731)、973计划项目(NO.2010CB529704)、高等学校博士学科点专项科研基金(NO.20069981001)、广州市科技计划项目(NO.2007J1-C0301)资助. （NO.81030055）
