狂犬病病毒aG株糖蛋白遗传稳定性及生物信息学分析

目的研究狂犬病病毒(rabies virus,RV)aG株糖蛋白(glycoprotein,GP)的遗传稳定性,并对GP基因组进行序列测定及生物信息学分析。方法采用RT-PCR法扩增6代次aG株RV(4aGV4、4aGV5、4aGV7、4aGV11、4a-GV15、4aGV18)G区基因,分别将产物克隆入pGEM-T载体中,测定序列并进行拼接;测定6代次aG株RV的滴度、荧光滴度和毒力;应用DNAStar和Mega4.0软件包中的相应软件对基因组序列进行分析,并与我国近期分离且具有地域代表性的RV街毒株进行基因同源性分析。结果 6代次aG株RV GP基因保持高度稳定,未出现核苷酸突变位点;6代次aG株RV的滴度有所不同,但其毒力指标基本一致;RV aG株与我国街毒流行株GP抗原区具有较高的同源性,且膜外区同源性远高于跨膜区及膜内区;aG株RV第147位关键位点氨基酸与街毒流行株该位点不同,其余各关键抗原位点在各毒株间均高度同源;6代次aG株RV关键毒力位点均未出现氨基酸突变,传代稳定,aG株RV与各街毒流行株关键毒力位点均高度同源;aG株病毒信号肽结构与我国多株流行株高度同源,而其GP则与法国巴斯德原株PV2061株及分离自美国的HEP-Flury株高度同源,与我国流行街毒株亦具有较高的同源性。结论 RV aG株GP遗传稳定,且与我国近期分离的街毒株高度同源,本研究为完善该毒株的质量控制及全面评价其在我国的适用性提供了实验依据。

Genetic stability and bioinformatics of glycoprotein of rabies virus aG strain

Objective To investigate the genetic stability of glycoprotein of rabies virus(RV)aG strain,and analyze the sequence and bioinformatics of GP genome.Methods GP genome sequences of aG strain of six passages,i.e.4aGV4,4aGV5,4aGV7,4aGV11,4aGV15 and 4aGV18,were amplified by RT-PCR and cloned into vector pGEM-T respectively,then sequenced and spliced.The aG strains of six passages were determined for titer,fluorescent titer and virulence,of which the GP genome sequences were analyzed by DNAStar and Mega4.0 software for homology to those of RV street virus strains isolated in China recently.Results The GP gene of aG strain of six passages were highly stable,in which no nucleotide mutation sites were observed.The RV titers of six passages were different,while the virulence indexes were basically in agreement.The GP antigenic domain of aG strain showed high homology to those of street virus strains in China,while the homology in extramembrane domain was significantly higher than those in transmembrane and intramembrane domains.The amino acid at key site 147 of RV aG strain was different from those of street virus strains,while the other key sites of various strains were highly homologous.No amino acid mutations were observed in key virulence sites of RV aG strain of six passages,indicating high genetic stability.The key virulence sites of aG strains were highly homologous to those of various street virus strains.The signal peptide structure of aG strain was highly homologous to those of various epidemic strains in China,while the GP was highly homologous to those of PV2061 strain isolated in France and HEP-Flury strain isolated in USA,and was homologous to those of street virus strains isolated in China.Conclusion The GP of RV aG strain showed high genetic stability,and was high homologous to those of street virus strains isolated in China recently,which provided an experimental basis for perfecting the quality control and complete evaluation of suitability of aG strain in China.