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柔嫩艾美耳球虫保守蛋白CHP559基因克隆与真核表达

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.07.024, PP. 161-168

Keywords: 柔嫩艾美耳球虫,CHP559基因,DF-1细胞,真核表达

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Abstract:

为构建柔嫩艾美耳球虫保守蛋白EtCHP559基因的真核表达重组质粒pcDNA3.1-flag-EtCHP559,并转染DF-1细胞进行表达。利用5'RACE技术克隆获得了柔嫩艾美耳球虫EtCHP559基因全长cDNA序列,该基因全长为1746bp,ORF为104-1327bp,编码407个氨基酸,编码蛋白的分子量约为46kD,并利用生物信息学分析该基因编码蛋白的性质、结构等特征,发现该蛋白含有跨膜结构和信号肽。通过RT-PCR扩增得到含完整开放阅读框的基因片段,并将其与真核表达载体pcDNA3.1-flag连接,构建了真核重组表达质粒pcDNA3.1-flag-EtCHP559,所构建的真核重组表达质粒pcDNA3.1-flag-EtCHP559经过PCR和双酶切鉴定,可见一条大小约为1224bp的目的条带;重组质粒转染DF-1细胞后,免疫印迹检测可见大小约为47kD的目的蛋白条带,间接免疫荧光实验显示在DF-1细胞中检测到绿色荧光。这些研究结果表明已成功获得了EtCHP559的全长序列,并成功构建了EtCHP559的真核重组表达质粒,在真核细胞DF-1中获得表达,为深入研究EtCHP559的功能奠定了基础。

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