【摘要】：目的構(gòu)建小鼠pMSV-Slfn5-GFP基因重組表達質(zhì)粒及基因結(jié)構(gòu)分析。方法提取小鼠肝臟組織中總RNA,反轉(zhuǎn)錄為cDNA。PCR擴增小鼠Slfn5編碼序列片段,并與pGEM-T Easy載體連接,連接產(chǎn)物轉(zhuǎn)化到大腸埃希菌DH5α中。挑取陽性克隆提取質(zhì)粒,經(jīng)限制性內(nèi)切酶HpaⅠ和XhoⅠ雙酶切鑒定。酶切鑒定正確的質(zhì)粒送Macrogen USA測序。測序正確的質(zhì)粒通過HindⅢ和XhoⅠ與pMSV-GFP連接,并命名為pMSV-Slfn5-GFP。通過UCSC(http://genome.ucsc.Edu/)分析小鼠Slfn5及其家族基因組結(jié)構(gòu),并通過NCBI確定Slfn5的蛋白結(jié)構(gòu)域。結(jié)果 Slfn5全長基因序列克隆到表達載體pMSV-GFP中,酶切鑒定片段大小為2.65kb。Slfn5的AAA_4蛋白結(jié)構(gòu)域由phylogeny.fr確定了該結(jié)構(gòu)在Slfn蛋白家族中的保守性。結(jié)論成功構(gòu)建了小鼠pMSV-Slfn5-GFP全長基因表達載體。
[Abstract]:Objective to construct the recombinant expression plasmid of mouse pMSV-Slfn5-GFP gene and analyze the gene structure. Methods Total RNA, was extracted from mouse liver tissue and reversely transcribed into cDNA.PCR to amplify mouse Slfn5 coding sequence, and then ligated with pGEM-T Easy vector. The ligation product was transformed into Escherichia coli DH5 偽. The positive clones were selected and identified by restriction endonuclease Hpa 鈪，

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