大片吸虫组织蛋白酶L全长序列的获取及其生物信息学分析

Isolation and Bioinformatic Analysis of Full-Length Catepsin L Gene

根据文库载体序列与原组织蛋白酶表达序列标签(EST)设计引物,以大片吸虫cDNA文库为模板,进行Touchdown PCR与梯度PCR相结合的扩增反应,扩增产物克隆入pMD18-T载体,经鉴定后测序,并采用生物信息学技术对序列进行开放阅读框(ORF)、编码氨基酸序列、蛋白质同源性比较、二级、三级结构等分析;结果所获序列全长990 bp,编码330个氨基酸,分子量36771.2 u,等电点5.44;具有较明显的螺旋、片层和无规卷曲等二级结构,α螺旋占17.3%,β折叠占27.0%,无规卷曲占55.8%;具有2个较明显的跨膜区和2个疏水区,未发现明显的信号肽和糖基化位点;氨基酸序列同源性比对显示其属于半胱氨酸家族;三级结构同源建模预测显示其与组织蛋白酶K(PDB number 2f7dA)有49%的一致性。

The full length cDNA sequence of Fasciola gigantica cathepsin L gene was isolated from cDNA library using the method which combined the touchdown and gradient PCR. The library vector and gene specific primer had been designed for performing the touchdown and gradient PCR. The products of PCR were cloned into pMD18-T vector and then sequenced. The bioinformatic analysis was used to predict the ORF, amino acid sequence, amino acid homology alignment, the secondary and tertiary domain, and so on. The extended sequence is composed of 990 base pairs coding 330 amino acids which MW and PI are 36 771.2 u and 5.44, respectively. The secondary structure prediction showed that the alpha helix,bata sheet and coil are 17.3%, 27.0% and 55.8%, respectively. Two transmembrane areas and two hydropathicity profiles were found but without significant signal peptide and glycosylation sites. Results of amino acid alignment against SWISS-Port database showed that the sequence belongs to the cysteine family. And the three diamensions picture has been gained by the way of Mock construction of molecular confirmation which showed the 49 % homology with the cathepsin K gene.