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乙型脑炎病毒(Japanese encephalitis virus,JEV)及其同血清群病毒在复制过程中存在核糖体-1移码表达NS1’蛋白的现象,但NS1’蛋白的功能目前仍不明确,核糖体移码在病毒复制过程中发挥的作用也没有清晰的认识。本研究通过分析乙型脑炎病毒基因组中核糖体移码序列,在对NS2A基因实施同义突变基础上,设计8对引物,通过PCR突变方法改变核糖体移码结构域移码效率或者病毒NS1’蛋白编码框长度。利用获得的突变毒株,测定分析不同突变对病毒NS1’蛋白表达情况、病毒复制能力和病毒毒力的影响。实验中分别将PCR突变片段替换JEV感染性克隆pJEHEN中相应片段,获得了8株突变全长克隆。经体外转录合成RNA,再以RNA转染BHK-21细胞,拯救出8株突变病毒vJETA、vJECT、 vJETC、vJESTC、vJESGA、vJE2R、vJET1和vJET2。通过RT-PCR扩增突变病毒中含突变位点的基因组片段,测序证实了vJETA株于3558bp处存在T-A突变；vJECT株于3555bp处存在C-T突变；、rfETC株于3561bp存在T-C突变；vJESTC株于3573bp处存在T-C突变；vJESGA株于3600bp处存在G-A突变；vJE2R株于3607bp-3609bp存在CGC-AGG突变；vJETl株于3690bp处存在T-A突变；vJET2株于3690bp和3819bp处存在T-A突变,突变碱基均位于各相应毒株设计突变位点。将突变病毒vJETA、vJECT、vJETC、vJESTC、vJESGA、vJE2R、vJET1和vJET2感染Vero细胞后,Western Blot检测显示,vJETC株、vJECT株和vJESTC株能检测到NS1蛋白和NS1’蛋白的表达；vJETA株和vJESGA株未能检测到NS1’蛋白的表达,只能检测到NS1蛋白的表达；vJE2R株、vJET1株和vJET2株均能检测到NS1蛋白的表达,同时vJE2R株能表达编码框缩短即蛋白分子量减小的NS1’蛋白,也能检测到vJET1株与vJET2株分别表达NS1’蛋白编码框不同程度延伸即蛋白分子量不同程度增加的NS1’蛋白。突变对病毒复制能力也造成了不同程度的影响,与亲本病毒vTHen相比,JE2R株与vJESGA株病毒复制能力与vTHen株基本一致,vJETC株与vJESTC株病毒复制能力有所下降；而突变毒株vJECT, vJETA, vJET1和VJET2与对照毒株相比,复制能力明显降低。但突变毒株复制能力与NS1’蛋白表达情况没有直接联系。通过动物实验发现,突变对病毒的毒力也造成了不同程度的影响。神经毒力试验中,vJE2R株与vJESGSA株与对照毒株相比基本一致,vJESTC株与vJETC株神经毒力有所下降,vJETA株、vJECT株、vJET1株和vJET2株神经毒力明显下降。神经侵袭力试验中,vJESGA株、vJE2R株和vJETC株与对照毒株相比基本一致,vJETA株、vJECT株、vJESTC株、vJET1株和vJET2株神经侵袭力明显下降。但突变毒株神经毒力和神经侵袭力与NS1’蛋白表达情况没有直接联系。通过获得实验结果可以发现,针对核糖体移码结构域进行突变操作能够影响NS1’蛋白的表达状况,但病毒的复制能力和毒力的改变与NS1’蛋白的表达情况并不存在直接联系,因此认为NS1’蛋白不具备直接影响病毒复制能力和毒力的功能。