口蹄疫病毒株AF72 VP3的结构模拟与分析

Construction and Analysis of VP3 from a Foot-and-mouth Disease Virus Strain AF72

摘要:

以口蹄疫病毒株AF72 RNA为模板, 反转录并扩增目的基因, PCR纯化产物与pGEM T- Easy载体连接并转化JM109菌株, 用凝胶电泳、PCR和SpeⅠ/ SphⅠ双酶切法鉴定为阳性的重组质粒进行测序。比对测序结果确定AF72 VP3的核苷酸序列, 利用同源建模的方法建立AF72 VP3结构蛋白的3D结构, 在此基础上, 综合亲水性、可塑性、抗原指数以及表面可能性等参数预测AF72 VP3结构蛋白的B细胞抗原表位。分析表明, 口蹄疫病毒VP1、VP2、VP3和VP4在核苷酸水平上的变异率是无差异的(P > 0.05); 而它们在氨基酸水平上的变异率差异显著(P < 0.05)。该毒株与20株源于GenBank中的VP3氨基酸序列比对发现其保守区主要位于第1~24、24~35、36~42、45~56、65~122、124~172、177~210、211~219位。AF72 VP3结构蛋白三维空间结构可分为A、B和C 3个结构区域, 蛋白呈现较规则的空间构象, 其中18~23、30~44、60~75、113~124、130~142、193~220氨基酸区段是AF72 VP3结构蛋白可能的B细胞抗原表位区域, 该结果将为进一步的FMDV多表位疫苗研究提供更有价值的参考信息。

Abstract:

RNAs isolated from the foot-and-mouth disease virus strain AF72 were used as templates for RT-PCR to amplify the target gene. The PCR products purified were cloned into the pGEM-T easy vectors and transformed into E. coli JM109. The positive recombinant plasmids were identified by electrophoresis, PCR, and restriction digestions with Spe I and Spe I analysis. The nucleotide sequences were confirmed by comparing with the full-length sequence of the other reference strains. The 3D model of structure protein VP3 of FMDV strain AF72 was obtained using homology modeling. On the basis of several parameters, such as hydrophilicity, flexibility, antigenic index, and surface probability, the B cell epitopes on VP3 were predicted. After analyzing the difference among VP1, VP2, VP3, VP4, at the nucleotide level, the mutation rates of these four encoding sequences were no difference (P > 0.05), however, at the amino acid level, those mutation rates were different (P < 0.05). The regions of 1th~24th、24th~35th、36th~42th、45th~56th、65th~122th、124th~172th、177th~210th、and 211th~219th in VP3 protein are most probably conservative. The 3D mold could be divided into A, B and C regions, the conformation of the VP3 of FMDV strain AF72 was regular, and the B-cell epitopes in VP3 probably exist in the following regions: 18 aa~23 aa, 30 aa~44 aa, 60 aa~75 aa, 113 aa~124 aa, 130 aa~142 aa, and 193 aa~220 aa. This result offers valuable information for design of FMDV multi-epitope vaccine against FMDV.