农业科学与技术：英文版
農業科學與技術：英文版
농업과학여기술：영문판
Agricultural Science ＆ Technology
2012年
6期
1194-1197
,共4页
Toll%样受体%牛%HEK293%细胞%脂膜酸%IL-8
Toll%樣受體%牛%HEK293%細胞%脂膜痠%IL-8
Toll%양수체%우%HEK293%세포%지막산%IL-8
Toll-like receptor%Bovine%HEK293 cells%Lipoteichoic acid%Interleukin-8
[目的]构建牛TLR2全长基因表达质粒,并在HEK293细胞中表达。[方法]利用RT-PCR技术克隆TLR2基因的全长编码区,连接到pMD18-Tsimplevector,再亚克隆到pEGFP-N1载体,得到包含TLR2基因全长的重组真核表达质粒。将重组质粒瞬时转染到HEK293细胞。流式细胞计数法和共聚焦显微镜法检测转染效率和表达蛋白在细胞中的定位;qRT-PCR法检测TLR2 mRNA在HEK293/boTLR2中的表达。最后,通过脂膜酸刺激HEK293/boTLR2细胞试验来分析TLR2蛋白的生物活性。[结果]成功克隆TLR2基因全长并连接到真核表达载体,并在HEK293细胞中表达。在LTA刺激的条件下,转染重组质粒的细胞比未转染的HEK293细胞分泌更多IL-8。[结论]本研究建立的细胞模式为TLR激动剂和拮抗剂的筛选提供快速有效的手段,有利于开展TLR激动剂和TLRs相互作用的研究。
[目的]構建牛TLR2全長基因錶達質粒,併在HEK293細胞中錶達。[方法]利用RT-PCR技術剋隆TLR2基因的全長編碼區,連接到pMD18-Tsimplevector,再亞剋隆到pEGFP-N1載體,得到包含TLR2基因全長的重組真覈錶達質粒。將重組質粒瞬時轉染到HEK293細胞。流式細胞計數法和共聚焦顯微鏡法檢測轉染效率和錶達蛋白在細胞中的定位;qRT-PCR法檢測TLR2 mRNA在HEK293/boTLR2中的錶達。最後,通過脂膜痠刺激HEK293/boTLR2細胞試驗來分析TLR2蛋白的生物活性。[結果]成功剋隆TLR2基因全長併連接到真覈錶達載體,併在HEK293細胞中錶達。在LTA刺激的條件下,轉染重組質粒的細胞比未轉染的HEK293細胞分泌更多IL-8。[結論]本研究建立的細胞模式為TLR激動劑和拮抗劑的篩選提供快速有效的手段,有利于開展TLR激動劑和TLRs相互作用的研究。
[목적]구건우TLR2전장기인표체질립,병재HEK293세포중표체。[방법]이용RT-PCR기술극륭TLR2기인적전장편마구,련접도pMD18-Tsimplevector,재아극륭도pEGFP-N1재체,득도포함TLR2기인전장적중조진핵표체질립。장중조질립순시전염도HEK293세포。류식세포계수법화공취초현미경법검측전염효솔화표체단백재세포중적정위;qRT-PCR법검측TLR2 mRNA재HEK293/boTLR2중적표체。최후,통과지막산자격HEK293/boTLR2세포시험래분석TLR2단백적생물활성。[결과]성공극륭TLR2기인전장병련접도진핵표체재체,병재HEK293세포중표체。재LTA자격적조건하,전염중조질립적세포비미전염적HEK293세포분비경다IL-8。[결론]본연구건립적세포모식위TLR격동제화길항제적사선제공쾌속유효적수단,유리우개전TLR격동제화TLRs상호작용적연구。
[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.
