Toll样受体2胞外域及其氨基端和羧基端片段的克隆与序列分析

目的：为研究TLR2胞外域在肽聚糖诱导的信号转导中的功能，构建TLR2胞外域及其氨基端和羧基端片段真核表达载体。方法：提取HL－60细胞总RNA，以RT－PCR方法获取了TLR2胞外域cDNA片段，将其与pGEM-T Easy载体连接，转化E.coli JM109,建立了TLR2胞外域的cDNA克隆；籍此，又相继克隆了胞外域的氨基端和羧基端片段，然后将这三个片段克隆到真核表达载体pcDNA3中。结果：序列分析表明，与GenBank中的人TLR2全长cDNA序列比较，仅4个碱基不同，同源性为0．998。结论：成功获得了HL－60细胞TLR2胞外域及其氨基端和羧基端片段的克隆。

Cloning and sequencing of extracellular domain and N-Terminal and C-Terminal fragments of Toll-like receptor 2

AIM To find out the role of the extracellular domain of TLR2 in the peptidoglycan induced signaling pathway, mammalian expression plasmids of the extracellular domain of TLR2 and its two fragments were constructed. METHODS The cDNA fragment encoding complete extracellular domain was isolated by semi nested RT PCR methodwith the total RNA extracted from HL 60 cells, linked into pGEM T Easy vector, transformed into E.coli JM109, and inserted into pcDNA3 vector. Then two fragments amplified from it were linked into pGEM T Easy vector and pcDNA3. RESULTS The result of sequencing indicated that the sequences of our cDNA was identical to the corresponding parts of the mRNA for human TLR2 in GenBank except 4 nucleotides, and with a homology of 0.998. CONCLUSION The extracellular domain of TLR2 and its two fragments are cloned successfully.