C57BL/6和A/J两品系小鼠脾脏消减cDNA文库的构建及差异基因分析

【目的】检测健康C57BL/6和A/J小鼠常规免疫指标，构建脾脏消减cDNA文库并筛选差异表达基因，以探讨两品系小鼠对猪链球菌抗病性差异的分子机制。【方法】利用ELISA法检测血清常规免疫指标，抑制性消减杂交（suppression subtraction hybridization，SSH）技术构建8周龄小鼠脾脏差异表达基因的cDNA文库。【结果】试验结果表明，A/J品系小鼠血清中IgA水平明显高于C57BL/6品系小鼠（P＜0.05），而血清IgG和IFN水平在两品系小鼠间均无显著差异。对筛选的149个阳性克隆进行测序，去除冗余的cDNA序列载体并聚类拼接后获得56条差异表达序列标签（ESTs）。利用Genebank的BLAST分析核酸和蛋白质同源性比较，26个不同的基因或ESTs具有高度的同源性，2条ESTs未找到同源序列。【结论】筛选到很多EST与信号转导、细胞凋亡及免疫等重要功能基因高度同源，为研究猪链球菌的致病机理和防治提供了实验依据。

Construction of Subtractive Hybridization cDNA Library to Screen Differentially Expressed Genes from the Spleen of C57BL/6 and A/J Mouse and Their Functional Analysis

【Objective】 The aim of this study was to discuss different mechanisms of infecting Streptococcus suis type 2 in C57BL/6 and A/J mouse strains by detecting the immunization parameters and constructed of cDNA library to screen different expressed genes from the spleen of C57BL/6 and A/J mouse strains.【Method】 Immunization parameters was detected by ELISA.The substracted cDNA library was constructed from the 8-week-old age spleen of C57BL/6 and A/J mouse strains by suppression subtraction hybridization(SSH).【Result】 The result showed that,the content of IgA was significantly higher in A/J mouse than C57BL/6 mouse,but there was no significantly differences in IgG and IFN between A/J and C57BL/6 mouse stains.A total of 149 positive clones were screened by PCR and sequenced.After dislodging,clustering and splicing the redundant cDNA sequence,total of 56 differentially expressed sequence tags(ESTs) were obtained in the whole subtractive cDNA library.After comparisons with GenBank using online software of the BLAST,total of 26 specific gene fragment and two unknow sequences were found.【Conclusion】 Many of those ESTs were highly homologous with the important functional genes with related to signal transduction,apoptosis and immunity and so on,and the results would provide useful baseline for screening and cloning specific resistant genes and understanding the molecular mechanism of Streptococcus suis.