正常猪外周血淋巴细胞cDNA文库构建及EST初步分析  

Construction of cDNA library from normal swine peripheral blood lymphocytes and preliminary analysis of its ESTs

作　　者：李晓宁[1] 孙石开[1] 蔡新斌[1] 杨坚[1] 苏丽娟[1] 章民[1] 李晓泉[1] 尹珊[1] 李延生[1] 罗廷荣[1] 

机构地区：[1]广西大学动物科学技术学院,南宁530005

出　　处：《南方农业学报》2011年第7期791-797,共7页Journal of Southern Agriculture

基　　金：国家"863"计划项目(2007AA10Z160);国家自然科学基金项目(31060336);广西科学研究与技术开发计划项目(桂科攻0480001);广西大学科学技术研究重点基金项目(2003ZD01)

摘　　要：【目的】构建猪淋巴细胞基因文库,初步绘制正常猪外周血淋巴细胞的基因表达谱,为进一步筛选免疫相关基因提供平台。【方法】以mRNA为模板,经反转录酶催化,在体外反转录成cDNA,与质粒载体连接后转化大肠杆菌,进一步扩增后,获得猪外周血淋巴细胞全部mRNA信息的cDNA克隆集合,并进行序列分析和注释。【结果】构建了正常猪外周血淋巴细胞cDNA文库,获得库容量为1.2×106PFU/mL、重组率为93.3%、85%插入片段处于750～2000bp的高质量文库;随机测定1100条ESTs,经拼接和聚类,获得152条高表达的基因重叠群(Contigs)和619条低表达基因——单拷贝的EST(Singletons);经序列比对分析,发现23.3%ESTs为与任何物种都没有匹配的新基因,75.9%ESTs为与猪没有匹配的新基因。用GO数据库对获得的EST进行基因功能分类和KEGG路径图分析,发现丝裂原活化蛋白激酶途径(Mi-togen-activatedproteinkinase,MAPK)、钙信号通路、胰岛素信号通路、脂肪细胞因子信号通路、Toll-like受体信号通路、B细胞受体信号通路和T细胞受体信号通路的基因均有较高表达。【结论】大部分的猪外周血淋巴细胞基因尚未被分离和鉴定出来,但这些基因mRNA转录和表达丰度均较高,且在猪外周血淋巴细胞中起着非常重要的作用。[Objective]The experiment was conducted to construct genomic library of swine lymphocytes and compare the gene expression profile of peripheral blood lymphocytes with normal swine for screening relative immunity gene. [Method]mRNA was used as the template and transcribed into eDNA using RT-PCR. After ligation to a plasmid vector, the eDNA was transformed to Escherichia coli and amplified. The eDNA clones of all swine peripheral blood lymphocytes mRNAs were obtained and their sequences were analyzed. [Result]A high-quality eDNA library was constructed from normal swine peripheral blood lymphoeytes. The eDNA library contained 1.2×10^6 PFU/mL, and showed 93.3% recombination rate with 85% inserted fragments ranging from 750 to 2000 bp. From this eDNA library, 1100 ESTs sequences were obtained by random sequencing, and 152 highly expressed genes （Contigs） and 619 less expressed genes （Singletons） were obtained through cluster analysis. The results of homology analysis revealed that 23.3% of the ESTs did not match with any species and correspond to new genes, and 75.9% of the ESTs represented new swine genes which did not match with any known swine gene sequences. According to gene functional classification and KEGG pathway analysis of EST using GO, it was found that the genes in mitogen-activated protein kinase, Ca information, insulinum, adipocyte factor, Toll-like receptor, B cell receptor and T cell receptor signal pathways were expressed highly. [Conclusion]Most of the genes in normal swine peripheral blood lymphocytes have not been isolated and identifieated yet, but the mRNA transcripts and expression abundance of those genes were higher and plays an important role in immunity.

关 键 词：猪 外周血淋巴细胞 CDNA文库 表达序列标签(EST) 

分 类 号：S828[农业科学—畜牧学] Q785[农业科学—畜牧兽医]