摘要
采用RT-PCR方法从小鼠巨噬细胞中扩增Toll样受体2(mTLR2)基因,得到基因全长CDS,经克隆测序正确后构建真核表达质粒pcDNA3.1-mTLR2。重组质粒瞬时转染HEK293T细胞,分别用RT-PCR与免疫荧光检测其表达及细胞定位。利用TLR2激活物pam3csk4刺激转染重组质粒的HEK293T细胞后,用双荧光素酶报告基因系统分析其下游转录因子NF-κB的转录活性。结果显示,mTLR2转染后成功表达并定位于细胞膜,pam3csk4刺激后荧光素酶活性显著高于生理盐水对照组,说明表达的mTLR2具有野生型分子的功能。本研究成功克隆与表达了mTLR2基因且表达产物具有生物学活性,为研究TLR2信号通路及其在抗寄生虫感染中的作用奠定了基础。
The Toll-like receptor 2（TLR2） gene was amplified from mouse macrophages by RT-PCR. After being cloned into the pMD18-T vector,TLR2 gene was subcloned into the pcDNA3.1 vector to con- struct the eukaryotic expression plasmid pcDNA3. 1-mTLR2. The recombinant plasmid was transiently transfected into the HEK293T cells,its expression and subcellular localization were analyzed by using RT- PCR and immunofluorescent assay,respectively. The results indicated that mTLR2 was successfully trans- fected,expressed,and localized at the cell membrane. TLR2 positive activator pam3csk4 was used to stimu- late the transfected HEK293T cells,dual luciferase report gene system was applied to measure the tran- scription activity of NF-~：B. The luciferase activity of the group stimulated by pam3csk4 was significantly higher than that of the normal saline control group,implying the recombinant mTLR2 had wild type function. In conclusion,the mTLR2 gene was successfully cloned and expressed and the protein had biological activity. This study provided the base for studving on TLR2 signal oathwav and its role in resistance to parasite infection.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第3期315-319,共5页
Chinese Veterinary Science
基金
"十一五"国家科技支撑计划重大项目(2010BAD04B01)
国家自然科学基金资助项目(31172312)
