釉成熟蛋白真核表达载体的构建及其在293T细胞中的表达

Construction of recombinant eukaryotic expression vector pcDNA3.1-Amelotin and expression in 293 T cells

目的:构建釉成熟蛋白真核表达载体pcDNA3.1-Amelotin,并观察其在293T细胞中的表达情况。方法:以出生后6 d的昆明小鼠下颌磨牙中提取的总RNA为模板,设计合成特异性引物,通过RT-PCR法扩增小鼠的釉成熟蛋白[Amelotin(GeneID:71421)],包括编码区全长在内的部分cDNA序列,克隆到pMD18-T克隆载体,酶切鉴定正确;然后亚克隆至pcDNA3.1真核表达载体中,双酶切鉴定及测序正确。将构建的重组载体磷酸钙法转染293T细胞,检测Amelotin的表达情况。结果:成功克隆了Amelotin基因编码区全长序列;构建出重组真核表达载体pcDNA3.1-Amelotin,磷酸钙法转染后可检测到Amelotin基因在293T细胞中的表达。结论:构建了重组pcDNA3.1-Amelotin真核表达载体,并成功表达,为进一步研究该蛋白的生物学活性奠定了基础。

AIM： To construct a novel recombinant eukaryotic expression vector peDNA3.1 - Amelotin and detect its expression in 293T cells. METHODS： Total RNA were extracted from the mandibular molar of mouse line Kunming. Amelotin gene was amplified by RT- PCR method,and then chined to pMD18 -T according to A -T. The gene was identified by restrictive enzyme,and then subcloned to vector by BamH I and Xho I restrictive enzyme digestion and ligation. Amelotin gene was transducted into 293T cells by Ca3 （PO4） 2 method. RESULTS ： pcDNA3. 1 - Amelotin recombinant vector was successfully constructed. It had a higher level expressed in 293T cells. CONCLU- SION：pcDNA3. 1 recombinant vector carrying Amelotin gene was constructed and Amelotin gene was successfully transfected into 293T cells. It will lay the basis for further study of this gene.