家蝇14-3-3蛋白基因的cDNA克隆与序列分析

cDNA Cloning and Bioinformatic Analysis of Musca domesitca 14-3-3

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 28(a+)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771 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。

The aim of this study is to clone the cDNA sequence of Musca domestica14-3-3（MD14-3-3）,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 MD143-3 was 771 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,containing many potential modified sites.The secondary structures were mainly composed of αcoiling and random coil.The gene coding for MD14-3-3 was amplified by polymerase chain reaction（PCR）,then the PCR product was transformed into the E.coliDH5α through being linked with pET 28a（＋）vector.As demonstrated by PCR,double enzyme digestion and DNA sequencing,it was confirmed that the recombinant expression plasmid was construction succeed.