人TLR2和TLR4胞外区cDNA的克隆  

Cloning of human cDNA of TLR2 and TLR4 extracellular domain

作　　者：孙守勋[1] 李强[1] 刘静[1] 白晓[1] 蒲晓允[1] 

机构地区：[1]第三军医大学附属新桥医院检验科,重庆400037

出　　处：《免疫学杂志》2008年第4期464-466,469,共4页Immunological Journal

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(2006B-2)资助

摘　　要：目的从脂多糖诱导的外周血单个核细胞克隆人Toll样受体2(TLR2)和Toll样受体4(TLR4)胞外区cDNA。方法用不同浓度脂多糖在不同时间刺激单个核细胞后提取总RNA,RT-PCR方法半定量测定TLR2和TLR4的表达,应用pUCm-T载体克隆胞外区cDNA,双酶切以及DNA测序进行鉴定。结果单个核细胞在四种浓度脂多糖刺激3h、6h和12h后,TLR2和TLR4表达并不相同。15μg/mL脂多糖作用6hTLR2表达最高,10μg/mL脂多糖作用3hTLR4表达最高。经RT-PCR扩增TLR2和TLR4胞外区cDNA分别为1700bp和1900bp,T载体克隆、酶切鉴定及测序分析后证实,目的片段与GenBank中序列一致。结论脂多糖的诱导对外周血单个核细胞TLR2和TLR4表达有显著影响,并且成功克隆了胞外区cDNA。Objective To clone human cDNA of TLR2 and TLR4 extracellular domain from peripheral blood mononuclear cells induced by lipopolysaccharide （LPS）. Methods The total RNA from mononuclear cells, which were affected by different doses and times with LPS, were extracted and the gene expression of TLR2 and TLR4 were measured by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. The gene was cloned by pUCm-T vector, and then digested and sequenced. Results After the mononuclear cells were stimulated with 4 different doses of LPS for 3 h, 6 h and 12 h, the expressions of TLR2 and TLR4 were different. The expression of TLR2 was reached to its peak after 6 h stimulation of 15 p.g/mL LPS, while the expression of TLR4 reached to its peak after 3 h stimulation of 10 μg/mL LPS. The fragments of 1 700 bp and 1 900 bp were amplified by RT-PCR. The cloning, enzyme digestion, and sequencing confirmed that the gene was consistent with standard sequence of GenBank. Conclusion The expressions of TLR2 and TLR4 are changed obviously after LPS induction. The cDNA of TLR2 and TLR4 extracellular domain are successfully cloned from peripheral blood mononuclear cells.

关 键 词：TLR2 TLR4 LPS 单个核细胞 胞外区 基因表达 

分 类 号：R392.11[医药卫生—免疫学]