山羊痘病毒069蛋白的表达与结构分析

Expression and structural analysis of goat pox virus 069 protein

为了分析山羊痘病毒THx株069基因的分子特征及其编码蛋白的生物学功能,本试验应用PCR技术对069基因进行扩增,并构建原核表达载体,经测序鉴定后用IPTG诱导表达其蛋白,应用SDS-PAGE和Western blot进行蛋白鉴定。用DNAStar及Mega软件对GenBank登录的24个该基因参考序列进行同源性比对并作遗传进化树分析;同时采用生物信息学分析方法对069蛋白信号肽、跨膜结构、糖基化位点、磷酸化位点、抗原决定簇、亲水性、二级及三级空间结构进行预测。结果表明:GTPV-069号基因全长为573 bp,与参考株GTPV-pellor相似度高达97.19%,经同源性比对与遗传进化树分析发现,山羊痘病毒THx株069基因序列与绵羊痘亲缘关系较近,在同一分支上,与疙瘩皮肤病和其他山羊痘亲缘关系较远。SDS-PAGE鉴定表明069蛋白大量表达于上清中,条带清晰。Western blot结果证明069蛋白与His标签确实进行了融合表达;生物信息学分析发现GTPV-069蛋白无信号肽、含有跨膜结构,跨膜区在29~51 aa之间,为典型的膜蛋白。069蛋白含有8个抗原决定簇、1个糖基化位点和14个磷酸化位点。二级结构分析显示,069蛋白中α螺旋、无规则卷曲、延伸链和β-转角分别占43.46%,25.65%,23.04%和7.85%,三级结构预测发现069蛋白以α螺旋为主。本试验为后期探究羊痘病毒其他部分生物学功能以及新型羊痘疫苗的研制提供了依据。

In order to analyze the molecular characteristics of goat pox virus THx strain 069 gene and the biological function of its encoding protein,PCR technology was used to amplify the 069 gene and construct prokaryotic expression vector.After sequencing and identification,IPTG was used to induce the expression of its protein.SDS-PAGE and Western blot were used to identify the protein.DNAStar and Mega software were used to compare the homology of 24 reference sequences from GenBank and to make phylogenetic tree analysis,respectively.Meanwhile,bioinformatics analysis was used to predict 069 protein signal peptide,transmembrane structure,glycosylation site,phosphorylation site,antigenic determinant cluster,hydrophilicity,secondary and tertiary spatial structure.The results showed that the total length of GTPV-069 gene was 573 bp,and the similarity with reference strain GTPV-pellor was 97.19%.By homology comparison and genetic evolutionary tree analysis,the sequence of THx strain 069 gene of goat pox virus was closely related to sheep pox,but far related to keloid skin disease and other goat pox on the same branch.SDS-PAGE showed that 069 protein was abundantly expressed with clear bands in the supernatant.Western blot results showed that 069 protein was indeed fused with His tag.Bioinformaticsanalysis showed that GTPV-069 protein had no signal peptide and transmembrane structure.The transmembrane region was between 29-51 aa,which was a typical membrane protein.069 protein contains 8 antigenic determinants,1 glycosylation site and 14 phosphorylation sites.Secondary structure analysis showed that 43.46%,25.65%,23.04% and 7.85% of the 069 protein were alpha helix,irregular crimp,elongation chain and beta-angle,respectively.Further prediction of the tertiary structure of069 protein showed that the 069 protein was dominated by alpha helix.This study provides a reference for further research on the biological functions of other parts of sheep pox virus and the development of a new type of sheep pox vaccine.