东方田鼠特异DNA片段的克隆及核苷酸序列分析  

The Cloning of Specific DNA Fragment in Microtus Fortis and Its Nucleotide Sequencing

作　　者：杨榕[1] 胡维新 朱敏[1] 彭兴华[1] 

机构地区：[1]中南大学湘雅医学院分子生物学研究中心,长沙410078

出　　处：《中国实验动物学报》2001年第2期67-72,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家科技部"九五"国家科技攻关项目! (96 A2 3 0 6 0 4 ) ;湖南省科技厅"九五"科技攻关配套基金;国家自然科学基金!(3 0 0 70 4

摘　　要：目的　获得东方田鼠的特异DNA序列。方法　Aβ基因使用PCR ,基因克隆 ,斑点杂交 ,DNA序列分析 ,生物信息学技术。结果　根据小鼠MHCⅡ外显子 2及其两侧序列 ,合成引物并扩增东方田鼠基因组DNA ,将PCR产物回收、测序后 ,分别设计内引物扩增东方田鼠基因组DNA ,其中一对引物可得到特异性扩增带 ,将得到的DNA片段插入PGEM Teasy载体 ,进行序列分析。用这对引物扩增人、昆明小鼠、BALB c小鼠及C57BL 6J小鼠基因组DNA ,均无扩增产物。以东方田鼠特异性扩增产物为探针进行斑点杂交 ,除东方田鼠基因组DNA外 ,其他几种动物基因组DNA均为阴性结果。进一步对该DNA片段进行了BLAST同源性搜索和外显子预测 ,在Genbank中没有发现高度同源序列 ,并且找到一个可能的外显子 ,该外显子由 69个氨基酸组成。结论　获得的DNA片段为东方田鼠的特异片段 ,这将为从分子水平深入研究东方田鼠的遗传背景、生物进化规律以及东方田鼠抗日本血吸虫的机理奠定基础。Objective\ To acquire specific DNA sequence of Microtus Fortis.Methods\ PCR,gene cloning,dot blot hybridization,DNA sequencing and bioinformatics analysis were used.Results\ A pair of primers were designed according to exon 2 of Aβ gene and its flanking sequences of mouse MHC-Ⅱ genes to amplify genomic DNA of Microtus Fortis,and non-specific products were generated.After sequencing of the PCR products,internal primers were designed to amplify genomic DNA of Microtus Fortis.Only one pair of the internal primers could generate a specific DNA fragment.This specific DNA fragment was sequenced after being cloned into T-easy vector.Genomic DNA of human,Kunming mouse,BALB/c mouse and C 57BL/6J mouse could not be amplified by this pair of primers to get the corresponding specific DNA fragment.Results of dot blot hybridization revealed that only genomic DNA of Microtus Fortis could be hybridized by this specific DNA fragment,while genomic DNA of other species could not.Furthermore,BLAST analysis and exon prediction were performed respectively,which revealed that there was no homologue in Genbank and a putative exon made up of 69 amino acids.Conclusion\ The acquired DNA fragment is specific to Microtus Fortis.All of these results lay a foundation at molecular level for further research work about genetic background and evolution of Microtus Fortis,as well as its mechanism of resistance to infection of Schistosoma japonicum.\;

关 键 词：东方田鼠 扩增 外显子 DNA片段 斑点杂交 引物 特异性 基因组DNA 动物基因组 生物进化 

分 类 号：Q78[生物学—分子生物学] R383.24[医药卫生—医学寄生虫学]