非复制型痘苗病毒修饰株NTV-C7L构建及毒力初步评价

Construction and virulence evaluation of nonreplicative vaccinia virus modified strain NTV-C7L

目的通过构建非复制型痘苗病毒修饰株NTV-C7L,在细胞和小鼠体内分别评价病毒毒力。方法通过同源重组构建非复制型痘苗病毒修饰株NTV-C7L,通过病毒的细胞扩散能力和复制能力,以及小鼠呼吸道感染实验和小鼠尾部划痕实验分别评估NTV-C7L的体外和体内毒力。结果经同源重组蓝白斑纯化获得的非复制型痘苗病毒修饰株NTV-C7L,通过PCR和Western blot鉴定证明C7L基因正确插入并能表达C7L蛋白;在人胚肺成纤维细胞(MRC-5)和小鼠胚胎成纤维细胞(BALB/C 3T3)中,NTV-C7L的扩散能力和复制能力比NTV强,但低于VTT;痘苗病毒小鼠滴鼻实验结果表明,106PFU病毒感染后TTV组小鼠体重与NTV及NTV-C7L组相比,体重下降差异具有统计学意义(t=6.56,P<0.001;t=5.73,P<0.001),NTV组与NTV-C7L组相比,体重下降差异无统计学意义(t=0.597,P=0.081);10^7PFU病毒感染后,NTV组及NTV-C7L组体重下降与TTV组相比,差异具有统计学意义(t=12.86,P<0.001;t=10.71,P<0.001)。NTV组与NTV-C7L组相比,体重下降差异具有统计学意义(t=4.616,P<0.001)。小鼠尾部皮肤划痕实验表明,接种10^6PFU VTT组小鼠在第5天尾部形成较为明显皮肤红肿、损伤,而10^7PFU NTV组和NTV-C7L组仅在局部形成红肿,且范围较VTT小,后逐渐结痂愈合。结论非复制型痘苗病毒修饰株NTV-C7L在细胞内及小鼠体内毒力较NTV升高,但明显低于VTT,为痘苗病毒载体优化及应用提供了参考数据。

Objective To evaluate the virulence of NTV modified strain NTV-C7L in vitro and in vivo.Methods The non-replicative vaccinia virus modified strain NTV-C7L was constructed by homologous recombination,in order to evaluate the virulence of NTV-C7L by cell diffusion ability and virus replication,intranasal challenge,and scratch of tail of mice.Results The homologous recombination non-replicating vaccinia virus modified strain NTV-C7L was selected by blue-white screening,the purity of NTV-C7L was identified by PCR and Western blot,the C7L gene was successfully inserted and expressed;In human embryonic lung fibroblasts(MRC-5)as well as mouse embryonic fibroblasts(BALB/C3T3),the replication ability of NTV-C7L was recovered compared with that of NTV,but was lower than that of VTT;the result of the intranasal route with 106 PFU vaccinia virus showed that the animals infected with TTV had slightly greater weight loss than NTV or NTV-C7L(t=6.56,P<0.001;t=5.73,P<0.001).Compared with the NTV-C7L group,the weight loss of the NTV group was not statistically significant(t=0.597,P=0.081);the intranasal route with 10^7 PFU vaccinia virus showed that the weight loss of the NTV group and NTV-C7L group was statistically significant compared with the TTV group(t=12.86,P<0.001;t=10.71,P<0.001).Compared with the NTV-C7L group,the weight loss of the NTV group was statistically significant(t=4.616,P<0.001);the result of skin scratch indicated that mice scarified with TTV(10^6 PFU)formed more obvious skin redness,swelling and injury on the post infected 5 days,whereas only redness was detected after scarification with a much higher dose(10^7)of NTV or NTV-C7L.Conclusions The intracellular and mouse virulence of NTV modified strain NTV-C7L was recovered compared with NTV,but was significantly lower than that of TTV,which provided reference for optimization and application of vaccinia virus vector.