摘要
为探究乙型脑炎病毒NS1′蛋白表达对小鼠神经毒力和神经侵袭力的影响,以乙脑病毒疫苗株SA14-14-2为模板,对NS2A基因进行A66G定点突变,并构建全长感染性克隆。体外转录后,将病毒RNA导入BHK21细胞,获得突变病毒SA14-14-2(A66G)。同样以乙脑病毒野毒株SA14为模板,采用相同方法构建并获得突变病毒SA14(G66A)。经测序和间接免疫荧光鉴定,证明突变病毒构建成功。Western blot检测发现疫苗株SA14-14-2和SA14(G66A)不表达NS1′蛋白,而野毒株SA14和SA14-14-2(A66G)能表达NS1′蛋白。生长曲线显示突变病毒的生长特性无明显改变。蚀斑实验发现SA14(G66A)的蚀斑较SA14更小,SA14-14-2(A66G)的蚀斑较SA14-14-2更大。细胞增殖活性实验显示NS1′蛋白表达对BHK21细胞的增殖有明显抑制作用(P<0.01)。疫苗株SA14-14-2与SA14-14-2(A66G)对1周龄小鼠脑内接种的LD50分别为297.9 PFU(0.02 mL)和143.4 PFU(0.02 mL),野毒株SA14和SA14(G66A)对3周龄小鼠脑内接种的LD50分别为0.8 PFU(0.03 mL)和2.3 PFU(0.03 mL)。疫苗株SA14-14-2与SA14-14-2(A66G)对2周龄小鼠腹腔接种的LD50分别为>5.65×10^(6)PFU(0.5 mL)和>1.46×10^(6)PFU(0.5 mL)。野毒株SA14和SA14(G66A)对3周龄小鼠腹腔接种的LD50分别为1.3×10^(3)PFU(0.5 mL)和1.2×10^(4)PFU(0.5 mL)。结果表明,乙脑病毒NS2A中第66位碱基是影响NS1′蛋白表达与否的关键位点,且NS1′蛋白表达能提高病毒对小鼠的神经毒力与神经侵袭力,其中对侵袭力的影响更为显著。
In order to probe deeply into the expression of Japanese encephalitis virus NS1'protein and its effect on neurovirulence and neuroinvasiveness in mouse,Japanese encephalitis virus vaccine strain SA14-14-2 was taken as the template,the A66G site directed mutation of NS2A gene was carried out,and full-length infectious clone was constructed.After in vitro transcription,the viral RNA was transferred into BHK21 cells,and obtained mutant virus SA14-14-2(A66G).Similarly,the mutant virus SA14(G66A)was constructed and obtained by taking the Japanese encephalitis virus wild strain SA14 as template.The mutant virus was successfully constructed after sequencing and indirect immunofluorescence identification.Western blot showed that Japanese encephalitis vaccine strains SA14-14-2 and SA14(G66A)did not express NS1'protein,but Japanese encephalitis wild strains SA14 and SA14-14-2(A66G)could express NS1'protein.Growth curves showed that no significant changes in the growth characteristics of the mutant viruses.The plaque experiment found that SA14(G66A)formed smaller plaque than SA14,and SA14-14-2(A66G)formed larger plaque than SA14-14-2.Cell proliferation activity assay showed that NS1'protein expression significantly inhibited the proliferation of BHK21 cells(P<0.01).The LD50 of vaccine strains SA14-14-2 and SA14-14-2(A66G)for intracerebral inoculation in 1-week-old mice were 297.9 PFU(0.02 mL)and 143.4 PFU(0.02 mL),and the LD50 of wild strains SA14 and SA14(G66A)for intracerebral inoculation in 3-week-old mice were 0.8 PFU(0.03 mL)and 2.3 PFU(0.03 mL)respectively.The LD50 of vaccine strains SA14-14-2 and SA14-14-2(A66G)for intraperitoneal inoculation in 2-week-old mice were>5.65×10^(6) PFU(0.5 mL)and>1.46×10^(6) PFU(0.5 mL)respectively,and the LD50 of wild strains SA14 and SA14(G66A)for intraperitoneal inoculation in 3-week-old mice were 1.3×10^(3) PFU(0.5 mL)and 1.2×10^(4) PFU(0.5 mL)respectively.The results of this study showed that whether the base at position 66 of Japanese encephalitis virus NS2A is the key site aff
作者
任阳
陈岚
杨俊杰
黄荣
冯亚岚
袁磊
杨健
REN Yang;CHEN Lan;YANG Jun-jie;HUANG Rong;FENG Ya-lan;YUAN Lei;YANG Jian(Coll.of Basic Med.&Forensic Med.,N.Sichuan Med.Coll.,Nanchong 637000;Jintang Count.1st People's Hosp.,W.China Hosp.Sichuan Uni.,Jintang Hosp.,Chengdu 610000)
出处
《微生物学杂志》
CAS
CSCD
2023年第3期86-98,共13页
Journal of Microbiology
基金
南充市川北医学院校地合作项目(20SXQT0239,20SXQT0338)。
