痘苗生长因子和胸苷激酶双基因缺失表达双荧光溶瘤痘病毒的构建及其对胰腺癌细胞的杀伤作用

［摘要］目的: 构建痘苗生长因子(vaccinia growth factor，VGF)和胸苷激酶(thymidine kinase，TK)双基因缺失同时表达绿色荧光蛋白（GFP）和DsRed双荧光的溶瘤痘病毒，并研究其对胰腺癌细胞的杀伤作用。方法: 在病毒TK基因处利用同源重组方法将病毒晚期启动子F17R调控的GFP基因和早晚期启动子SEL调控的DsRed基因克隆入缺失VGF基因的VSC20亲本病毒，构建同时缺失VGF和TK双基因的重组溶瘤痘病毒vvDD-GFP DsRed。同时利用CCK 8法和结晶紫染色法检测该病毒对胰腺癌细胞Patu8988和BxPC-3的杀伤作用。结果： 通过同源重组和软琼脂筛选获得vvDD-GFP-DsRed重组溶瘤痘病毒。CCK 8法结果显示，与MOI=0相比，MOI分别为1，10时，vvDD-GFP-DsRed对Patu8988细胞和BxPC-3细胞具有明显的杀伤作用，且在此范围内随MOI值增加杀伤效果逐渐增强(P均＜0.05)。与MOI=0相比，Patu8988细胞在MOI为0.01时存活率低(P＜0.05)，且在结晶紫染色法测定中形成了明显的空斑。结论： 成功构建vvDD GFP DsRed重组溶瘤痘病毒，其对胰腺癌细胞具有明显的杀伤作用。

Construction of VGF and TK gene deletion expressing double fluorescent oncolytic poxviruses and its killing effect on pancreatic cancer cells

Abstract]Objective: To construct double fluorescent oncolytic vaccinia virus expressing both GFP and DsRed with the dual deletion of vaccinia growth factor(VGF) and thymidine kinase(TK), and to explore the effect of virus on the cell lysis of pancreatic cancer cells in vitro. Methods: GFP regulated by the viral late promoter and DsRed controlled by early and late promoter SEL were inserted into viral TK locus by homologous recombination with parental viral VSC20, which was deleted by VGF gene. The anti cancer effect of vvDD GFP DsRed was tested by CCK 8 and crystal violet staining in vitro. Results: Recombinant vvDD GFP DsRed was got by homologous recombination and purified by soft agarose. CCK 8 detection results showed that compared with MOI=0, when MOI=1,10 respectively, vvDD GFP DsRed had stronger lytic effect on Patu8988 and BxPC 3 cells, and the lytic effect gradually increased among the range with MOI value increasing(P＜0.05). Compared with MOI=0, the survival rate of Patu8988 cells was lower when MOI=0.01(P＜0.05); and in the crystal violet staining method the plaque could obviously be seen. Conclusion: vvDD GFP DsRed recombinant oncolytic poxvirus was successfully constructed, which is able to kill pancreatic cancer cells in vitro.