小鼠pMSV-Slfn5-GFP表达质粒构建及基因结构分析

Construction of pMSV-Slfn5-GFP plasmid and analysis of gene structure in mice

目的构建小鼠pMSV-Slfn5-GFP基因重组表达质粒及基因结构分析。方法提取小鼠肝脏组织中总RNA,反转录为cDNA。PCR扩增小鼠Slfn5编码序列片段,并与pGEM-T Easy载体连接,连接产物转化到大肠埃希菌DH5α中。挑取阳性克隆提取质粒,经限制性内切酶HpaⅠ和XhoⅠ双酶切鉴定。酶切鉴定正确的质粒送Macrogen USA测序。测序正确的质粒通过HindⅢ和XhoⅠ与pMSV-GFP连接,并命名为pMSV-Slfn5-GFP。通过UCSC(http://genome.ucsc.Edu/)分析小鼠Slfn5及其家族基因组结构,并通过NCBI确定Slfn5的蛋白结构域。结果 Slfn5全长基因序列克隆到表达载体pMSV-GFP中,酶切鉴定片段大小为2.65kb。Slfn5的AAA_4蛋白结构域由phylogeny.fr确定了该结构在Slfn蛋白家族中的保守性。结论成功构建了小鼠pMSV-Slfn5-GFP全长基因表达载体。

Objective To perform mouse pMSV-Slfn5-GFP gene recombinant expression plasmid construction and gene structure analysis. Methods Total RNA was extracted from mouse liver and turned into cDNA by reverse transcription. Mouse Slfn5 coding sequence （CDS） fragment was amplified by PCR and connected with the pGEM-T Easy vector. The connected product was transferred the E. coli DHSa. The positive clones were selected for extracting plasmid, which was identified by double enzyme of restriction endonuclease Hpa I and Xho I . Then correct plasmid identified by enzyme digestion was sequenced by Macrogen USA. Then correct plasmid by sequencing was connected with pMSV-GFP by HindHI and Xbo I ,which was named as pMSV-Slfn5-GF. UCSC （http：//genome. ucsc. Edu/） was used to analyze mouse Slfn5 and its family genomic structure. Slfn5 protein structural do- main was determined by NCBI. Results Slfn5 full-length gene sequence was cloned into the expression vector pMSV-GFP,the frag- ment size was about 2.65 kb by enzyme digestion identification. The conservatism of AAA_4 protein domain in Slfn5 protein family was determined by phylogeny, ft. Conclusion Mouse full-length gene pMSV-Slfn5-GFP expression vector is successfully constructed.