本文選題：CYPB　切入點(diǎn)：載體構(gòu)建　出處：《中國(guó)獸醫(yī)學(xué)報(bào)》2017年09期

【摘要】：CYP26B1是調(diào)節(jié)體內(nèi)視黃酸(retinoic acid,RA)水平的一個(gè)關(guān)鍵酶,廣泛存在于哺乳動(dòng)物器官和組織中,對(duì)軟骨細(xì)胞的分化具有重要的調(diào)節(jié)作用。本試驗(yàn)參照NCBI GenBank中已發(fā)表的牛CYP26B1基因序列,設(shè)計(jì)特異性引物。利用PCR方法擴(kuò)增CYP26B1基因的編碼區(qū),并與pGEM-T載體連接,經(jīng)EcoRⅠ和XhoⅠ雙酶切后與pcDNA3.1載體融合得到CYP26B1過(guò)表達(dá)重組質(zhì)粒。經(jīng)測(cè)序鑒定后,轉(zhuǎn)染鹿茸軟骨細(xì)胞,利用實(shí)時(shí)熒光定量PCR方法檢測(cè)CYP26B1mRNA表達(dá)變化。應(yīng)用MEGA軟件的Test Neighbor-Joining Tree法繪制CYP26B1基因系統(tǒng)進(jìn)化樹(shù)并進(jìn)行比對(duì)分析。結(jié)果顯示,重組質(zhì)粒pcDNA3.1-CYP26B1轉(zhuǎn)染鹿茸軟骨細(xì)胞后,CYP26B1的表達(dá)量顯著升高(P0.05)。進(jìn)化樹(shù)分析結(jié)果表明,梅花鹿CYP26B1基因與牛的基因同源性最高。本研究成功構(gòu)建了梅花鹿CYP26B1過(guò)表達(dá)載體,為進(jìn)一步研究CYP26B1在鹿茸生長(zhǎng)及再生中的作用奠定基礎(chǔ)。
[Abstract]:CYP26B1 is a key enzyme in regulating retinoic acid (retinoic acid) in vivo. It is widely found in mammalian organs and tissues and plays an important role in regulating the differentiation of chondrocytes. This study refers to the sequence of bovine CYP26B1 gene published in NCBI GenBank. Specific primers were designed. The coding region of CYP26B1 gene was amplified by PCR and ligated with pGEM-T vector. The recombinant plasmid of CYP26B1 overexpression was obtained by double enzyme digestion of EcoR 鈪，

本文編號(hào)：1687477