摘要
目的为研究巨噬细胞(Macrophage,Mφ)细胞在形成破骨细胞时细胞膜膜蛋白的表达及相互作用机制。方法C57BL/6小鼠长骨骨髓单个核细胞加入M-CSF和RANKL诱导形成破骨细胞,在Mφ细胞开始融合至形成破骨细胞的不同阶段开始收集细胞,提取总RNA,用CloneMiner TM cDNA文库构建试剂盒,采用Gateway非放射标记方法构建小鼠破骨细胞前体细胞全长cDNA文库及酵母表达文库,并将其转化酵母,观察其在酵母中的表达情况。结果构建的小鼠骨髓破骨细胞前体细胞cDNA入门文库滴度为6×106cfu/ml,库容量为3.5×107cfu,重组率为100%,重组子插入cDNA平均片段大小约为1.76kb。利用此入门文库构建的酵母表达文库,重组率100%,插入片段平均长度为1.82kb,绝大多数(87.5%)插入片段在1kb以上,酵母转化实验证实其可在酵母中完整表达。结论 Gateway非放射标记法构建的小鼠骨髓破骨细胞前体细胞cDNA入门文库和酵母表达文库均符合高质量文库要求,且可避免放射性物质的接触。
Objective To investigate membrane protein expression and its interaction in the course of macrophage（Mφ） fusion to form osteoclast cells.Methods Bone marrow cells from C57BL/6 mice were collected and the adherent cells were removed by suspending cells overnight on treated plate.Macrophage was induced with M-CSF.Osteoclast precursor cells were collected at different time point（every 4h）from cells beginning fusion till most cells becoming osteoclasts after the macrophage treated with RANKL.Total RNA was extracted from the osteoclast precursor cells.The cDNA entry library and the yeast expression library were constructed in succession by using Clone Miner TM cDNA construction kit non-radiolabeling technology.The yeast expression library was transfered into yeast and its expression in yeast was confirmed.Results The cDNA entry library was constructed with a titer of 6×106cfu/ml,total clones of 3.5×107cfu,with the percentage of recombinant clones about 100%.The average size of the insert was about 1.76kb.The yeast expression cDNA library was constructed subsequently by using the cDNA entry library with a titer of 0.94×106,total clones of 0.9×107,a mean insert size about 1.82kb and the percentage of recombinant clones was 100%.A preliminary experiment confirmed that the yeast expression library can express fully in yeast.Conclusion The yeast expression library constructed with non-radio labeling technology,which avoid radiation exposure,was good enough to meets the requirement of a standard cDNA library,thereby can be used in further study.
出处
《中国热带医学》
CAS
2011年第11期1323-1326,共4页
China Tropical Medicine
基金
广州市医药卫生科技重点项目(No.201102A212025)
关键词
小鼠
破骨细胞
cDNA质粒文库
构建
鉴定
Mice
Osteoclast precursor cell
cDNA plasmid library
Construction
Identification
作者简介
张玉平，男（1968～），副主任医师，博士，主要从事血液病研究。 通讯作者：E-mail：jnzhangyuping@163．com
