Wistar大鼠c-ski基因部分序列的克隆、测序及实时荧光定量PCR的建立  

Cloning and sequence analysis of partial c-ski gene of Wistar rat,and development of SYBR Green-based real-time fluorescence quantitative PCR

作　　者：李平[1] 刘苹[1] 熊仁平[1] 赵艳[1] 朱敏[1] 周元国[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所分子生物学中心,创伤、烧伤与复合伤国家重点实验室,重庆400042

出　　处：《解放军医学杂志》2009年第10期1221-1224,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30801195);国家重点基础研究发展计划(973计划)资助项目(G1999054205,2005cb522602)

摘　　要：目的测定Wistar大鼠c-ski基因部分序列,并应用于检测大鼠c-ski基因表达的实时荧光定量PCR的建立。方法提取培养的Wistar大鼠皮肤成纤维细胞总RNA,通过RT-PCR扩增,将扩增产物克隆到PMD-18T载体上并测序。根据获得的c-ski基因部分序列设计实时荧光定量PCR引物,建立SYBRgreen定量PCR检测方法,并检测培养的大鼠皮肤成纤维细胞c-skimRNA表达水平。结果获得的Wistar大鼠c-ski基因cDNA序列长561bp,为未曾报道的新序列,其与人、小鼠具有较高的同源性,Gen-Bank收录(收入号DQ409171)。建立的SYBRgreen定量PCR检测标准品在103～109拷贝范围内的相关系数为0.99149。体外培养的Wistar大鼠皮肤成纤维细胞样品中c-skimRNA水平为(1.02±0.18)×106拷贝/μl。结论新获得了Wistar大鼠c-ski基因部分序列,该序列可用于实时荧光定量PCR的建立。Objective To clone and analyze the partial c-ski gene of Wistar rat,and to develop SYBR Green-based real-time fluorescence quantitative PCR.Methods Total RNA was extracted from the cultured skin fibroblasts of Wistar rat,the partial c-ski gene was then amplified by reverse transcription polymerase chain reaction（RT-PCR） and cloned into eukaryotic expression vector PMD-18 T,then sequenced with Beckman CEQTM8000.The new partial sequence was used to design the oligodoxynucleotide primers,and the real-time quantitative fluorescence PCR was based on SYBR Green,which was obtained to examine the expression of c-ski mRNA in samples of the cultured skin fibroblasts of Wistar rat.Results The partial c-ski gene of Wistar rat obtained by RT-PCR and cloned into eukaryotic expression vector PMD-18 T was 561 bp in length,and shared high homology with that of human and mouse,and accepted by GenBank as new sequence（ACCESSION DQ409171）.The established real-time PCR showed high correlations（r=0.99149） with the standard curves from 10%3 to 10^9 copies.Conclusion The SYBR Green based real-time fluorescence PCR has been established successfully and can be used for determination of c-ski mRNA of skin tissue and cultured cells.

关 键 词：c-ski 克隆 分子 序列分析 聚合酶链反应 

分 类 号：R349.8[医药卫生—基础医学]