家蚕30kDa载脂蛋白19G1前体的生物信息学分析及克隆表达

Bioinformatic Analyses,Cloning and Expression of 30kDa Lipoprotein 19G1 Precursor from Bombyx mori

从本实验室构建的家蚕蛹cDNA文库中发现了一条编码低分子量30 kDa载脂蛋白19G1前体(lowmolecular mass 30 kDa lipoprotein 19G1 precursor)的EST序列(GeneBank登录号:AY568957),在NCBI数据库中比对后得知该基因的ORF长为771 bp,与基因组比对后发现该基因的ORF不含内含子,由单一外显子编码256个氨基酸残基的蛋白质,该蛋白属于家蚕30K蛋白家族,预测分子量约为29.5 kDa,等点电为7.29;信号肽分析显示该蛋白很可能存在信号肽。根据该基因ORF进行引物设计,以家蚕基因组为模板,PCR扩增获得该基因,将其克隆到pET-28a(+)载体中并导入大肠杆菌BL21(DE3),IPTG诱导表达,裂解菌作SDS-PAGE分析,结果表明该蛋白前体成功表达,为研究其功能打下了基础。

The EST of low molecular mass 30 kDa lipoprotein 19G1 precursor（GeneBank accession no. AY568957） is obtained by analyzing our cDNA bank of silkworm pupae. By means of bioinformatic methods, the ORF contains no intron but an exon of 771 bp encoding 256 amino acids; the predicted molecular weight is 29.5 kDa and the pI is 7. 29. Moreover, there is a signal peptide in this protein precursor. This apoprotein precursor gene is amplified from genomic DNA of Bombyx mori, cloned into pET-28a（＋） vector. The result of SDS-PAGE shows that the fusion protein is expressed successfully in E. coli BL21 （DE3） induced by IPTG, supporting for functional studies of this gene.