家蝇14-3-3蛋白基因的cDNA克隆及序列分析.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 年第 期 2013 9 家蝇 14-3-3 蛋白基因的 cDNA 克隆与序列分析 国果  吴沁怡  吴建伟  付萍  张勇 （贵阳医学院寄生虫学教研室，贵阳 550004） 摘 要 ： 从家蝇cDNA 文库中筛选获得家蝇14-3-3 （MD14-3-3 ）基因DNA 序列，以该基因的cDNA 文库质粒为模板，PCR 方法对MD14-3-3 基因进行扩增，以pET 28a（+ ）为载体构建重组质粒。采用生物信息学方法对该基因及其编码蛋白的基本理化性质、 疏水性、信号肽、二级结构和亚细胞定位等方面进行预测和分析。结果表明，MD14-3-3 基因ORF 全长77 1 bp ，编码257 个氨基酸， 理论分子量29.35 kD ；等电点5.78 ，属于亲水性的酸性蛋白，含有多种酶的结合位点，有14-3-3 家族结构域和活性位点，定位于 细胞核中，二级结构以α螺旋和无规则卷曲为主。经PCR、双酶切及NDA 测序结果均表明pET 28a （+ ）- MD14-3-3 重组质粒构建 成功。 关键词 ： 家蝇 14-3-3 基因克隆 生物信息学分析 - - cDNA Cloning and Bioinformatic Analysis of Musca domesitca 14 3 3 Guo Guo Wu Qinyi Wu Jianwei Fu Ping Zhang Yong （Dep artment of Parasitology ，Guiyan Medical College ，Guiyang 550004 ） Abstract:  The aim of this study is to clone the cDNA sequence of Musca domestica 14-3-3 （MD 14-3-3 ），analysis the gene and encoded protein sequnece of MD14-3-3 by the bioinformatics methods in the following aspects, such as general physical and chemical properties, hydrophobicity, signal peptide, secondary structure and subcellular localization. Results showed that the open reading frame of the MD14- 3-3 was 77 1 bp that encoded a putative protein with 257 amino acids. The protein, with predicted molecular weight 29.35 kD and pI of 5.78, had one active site of family 14-3-3, without signal peptide. It was a hydrophilicity acidic protein which was mainly located in cell nucleus possible,