人骨质疏松cDNA表达文库的构建及分析

Construction and Characterization o fa cDNA Expression Library from Human Osteporosis Tissue

目的构建人骨质疏松骨组织cDNA文库,为筛选与骨质疏松的发生特异相关的基因及探讨骨质疏松发病机制奠定基础。方法以Trizol一步法从骨质疏松小鼠长骨中提取总RNA,分离纯化mRNA,LD-PCR法反转录合成双链cDNA,以λTripEX2为载体,构建骨质疏松小鼠cDNA文库,对随机挑取的噬菌斑进行PCR鉴定。结果原始文库的滴度为4.8×105pfu/ml,总克隆数为4.6×105,重组率为96.2%,扩增后文库总滴度为6.1×109pfu/ml,插入片段多分布在0.5～2.6kb之间,绝大部分在1.4～1.9kb左右,文库质量鉴定结果表明,构建的骨质疏松小鼠cDNA文库具有较好的库容量、较高的重组率以及较大的插入片段。结论构建cDNA文库为克隆骨质疏松相关功能基因的研究奠定了基础。

Objective To construct a eDNA plasmid library from human osteporsis. Methods Total RNA was isolated from human osteporosis tissue using Trizol single-step method, and the mRNA was purified using mRNA purification kit. The firest-strabd eDNA were synthesized by SAART technique. Long distance PeR was then used to synthesized the double-strand eDNA that was used then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The eDNA fragments were ligated to λ TripEX2 vector and recombinant bacteriophages were packaged. The eDNA library was tittered and amplified using XL1-Blue as receptor bacterium. Thirteen plaques were randomly picked and rested using PeR with universal primer derived from the sequence flanking the vector. Results The titer of the -original library was 4.8× 10^6 pfu/ml with a recombinant rate of 97.6% and 4.6 ×10^6 clones in total , the amplified titer was 6.1× 10^9 pfu/ml. PCR amplification suggested the inserted cDNA fragments ranged from 0. 5 kb to 2.3 kb , mostly from 1.4 kb to 1. 9 kb. Conclusion The data indicate that the human osteporosis tissue eDNA library has high titer, high recombinant percentage and large inserted fragments. The construction of cDNA library has built a base for the functional 8enomic research of human osteporosis tissue .