A组轮状病毒多个结构蛋白抗原表位的串联表达及其免疫原性的检测

Linked Multi-epitopes of Several Rotavirus Structural Proteins as Antigens

从北京腹泻婴儿粪便提取的轮状病毒(rotavirus,RV)(T114株)的RNA中,克隆到轮状病毒结构蛋白基因vp4,vp6和vp7的全长cDNA,对它们编码的蛋白质序列和可能的抗原表位肽进行了预测,选择了RV主要抗原蛋白VP7、VP6和VP4的4个抗原表位肽,通过人工合成DNA的方式将这些抗原表位肽基因串联融合成一个阅读框RME(rotavirus multipleepitopes,RME)并构建原核表达载体.大肠杆菌表达的RME在ELISA反应中可被RV多克隆抗体识别,纯化的RME蛋白注射免疫小鼠可诱导特异性免疫应答,产生高滴度的同源氨基酸序列特异抗体和人RV抗体,其中针对RME的IgG抗体滴度达到l∶40 000,针对单个抗原表位EV7、EV6和EV4的IgG抗体滴度达l∶10 000～l∶20 000,针对RV Wa株的IgG抗体滴度较低为l∶2 500,但能特异地中和该病毒对MAC145细胞的侵染.上述结果为新型RV基因工程疫苗的研发提供了论据和基础.

The full length cDNAs of rotavirus structural protein genes,vp4,vp6 and vp7 were cloned from the rotavirus infected child stool specimen in Beijing through RT-PCR.The protein sequences and their antigenic determinants were predicted.According to the epitope peptide sequences,4 epitopes from these structural proteins were chosen,a DNA fragment encoding all these epitopes was synthesized and cloned into the prokaryotic expression vector.Multiple epitope protein（rotavirus multiple epitopes,RME） expressed in E.coli can be recognized by the polyclonal antibody of rotavirus,and induce immune response in mice.The specific antibody IgG induced by RME can recognize human rotavirus（Wa strain）,RME itself as well as individual epitope peptides.The antibody titer of IgG to RME is high（1∶40 000）,while the titers to EV4,EV6 or EV7 are in a range of 1∶10 000 to 1∶20 000.However the IgG titer to the Wa strain is lower,i.e.,1∶2 500.Intriguingly,the RME-induced IgG can neutralize the Wa strain rotavirus challenge in the MAC145 cell line.This research has laid the foundations of producing effective bioengineering vaccines to rotavirus.