口蹄疫病毒株OA/58 L蛋白酶的结构构建和功能分析

以口蹄疫病毒株OA/58 RNA为模板,反转录并扩增目的cDNA,然后与pGEM-T Easy载体连接并转化JM109菌株,提取的重组质粒用凝胶电泳、PCR和EcoR I酶切法鉴定。该毒株与A12,O1K,O1Campos和TW45/97毒株序列通过对比分析发现,L基因中有2个起始密码子,第二个是首选;并且确定氨基酸保守区主要位于第35～39,43～54,65～67,75～80,90～111,113～142,144～146,148～157,159～172和176～187位。L蛋白酶含有球状区域,碳端有一柔性杆状结构。合成的L蛋白酶可形成二聚体结构。第144位的Lys、148位的His和163位的Asp可能是L蛋白酶的活性位点。保守区氨基酸残基在维持蛋白的空间构像和功能方面具有重要作用。由拉马钱德兰图可证明本试验构建L蛋白酶的空间结构是合理的,它对于进一步的研究具有指导性。

Construction and Analysis of L Protease Structure and Function from a Foot-and-month Disease Virus Strain OA/58

Foot-and-mouth disease virus strain OA/58 RNAs were used as templates for RT-PCR.The amplified cDNA products were cloned into pGEM-T Easy vectors and transformed into JM109.The recombinant plasmids were identified by electrophoresis,PCR,and EcoR I cleavage.The nucleotide and amino acid sequences were compared with the L genes of the other 4 reference strains.The results show that between 2 initiation codons,there is a special function sequence which enables small subunit of ribosome to recognize and utilize the second AUG to translate L protease that is called for Lb;the regions of 35-39th,43-54th,65-67th,75-80th,90-111th,113-142th,144-146th,148-157th,159-172th and 176-187th in L protease most probably are conservative.By homology modeling the FMDV strain OA/58 L protease,the 3D mold of this protease was obtained.Resolution of the 3D structure of L protease showed a compact globular form with a flexible C terminal extension from 187th to 201th.Depending on the region of hydrophilicity residues forming hydrophilicity power,L proteases can form dimmers.Lys144,His148 and Asp163 may be active amino acids which form the active site of L protease.By Ramachandran Plot showing,3D mold of a FMDV strain OA/58 L protease is rather reasonable,this research should be used as an instruction in order to direct the work on FMDV L protease.