人胎儿骨膜全长cDNA文库的构建及测序

Library Construction and Large-Scale Sequencing of Human Fetal Periosteum Full-length cDNA

目的构建人胎儿的骨膜组织的全长cDNA文库,了解该组织表达谱的特征。方法以胎儿胫骨骨膜为材料,运用末端模板转换技术(SMART)构建cDNA文库,测定库容量并应用菌落PCR的方法确定平均插入片段长度,随后大规模测序并进行生物信息学处理分析。结果所构建的cDNA文库的库容量为5.2×106,平均插入片段的长度为1.2kb。随机测序了25000余个克隆的序列,拼接后为5230个聚类群(contigs)和4714个独立EST(singleton),Blast比对分析得到骨膜基因表达谱的概况,发现了23个候选的新基因。结论构建的骨膜组织的cDNA文库质量满足后续工作的需要,骨膜组织基因表达谱及一些新基因的获得为今后寻找骨膜组织特异性的基因从而研究骨相关疾病的发病机制、发现具有药物开发靶点的功能基因奠定了基础。

Objective To construct the library of human fetal periosteum full-length cDNA and examine the expression profile of the tissue. Methods By using the technique of Switch mechanism at 5＇end of mRNA template （SMART）, human fetal tibia periosteum was used to construct its fulllength library. The library was tittered, the average exogenous inserts of the recombinants were determined by use of clone-based PCR, and then colons in the library were sequenced on large scale basis. Subsequently, all sequences were annotated according to their DNA sequence homologies against the NCBI database by using the BLAST program. Results The titter of the cDNA library was 5.2 × 10^6, and the average insert length was 1.2 kb. More than 25 000 clones were sequenced, and 9944 non-redundant cDNA sequences were formed including 5 230 contigs and 4 714 singletons after initial alignment. BLAST showed the classification of gene groups expressed in perlosteum tissue and 23 new candidate genes were identified. Conclusions The quality of this full-length library can satisfy future study needs. The expression profile of human fetal periosteum and new genes identified lay a foundation for the elucidation of mechanisms of bone-related diseases and new target genes for gene therapy.