重组溶瘤痘病毒rVV-CCL5-SSTR2-Luc+的构建及其杀瘤活性

Construction of three strains of recombinantoncolytic vaccinia virus and their anti-tumor activity

目的在体外构建携带CCL5和SSTR2双基因的重组溶瘤痘病毒,并初步检测其溶瘤活性。方法利用基因工程策略,分步构建重组痘病毒真核表达质粒pSC65-CCL5-SSTR2-Luc+;经同源重组痘病毒Western Reverse(WR)株和23轮筛选纯化后,获得纯化的重组溶瘤痘病毒rVV-CCL5-SSTR2-Luc+;经扩大培养及浓缩纯化后获得高滴度的重组溶瘤痘病毒,用ELISA和Western blot分别验证感染重组溶瘤痘病毒的HeLa细胞CCL5和SSTR2的表达变化;用CCK-8法检测了rVV-CCL5-SSTR2-Luc+对两株肿瘤细胞系的杀伤活力。结果成功构建了重组痘病毒真核表达质粒pSC65-CCL5-SSTR2-Luc+;经同源重组、筛选纯化和浓缩后,获得滴度为9.4×10^8 PFU/mL的重组溶瘤痘病毒rVV-CCL5-SSTR2-Luc+;ELISA和Western blot结果表明,HeLa细胞感染rVV-CCL5-SSTR2-Luc+后,其CCL5和SSTR2蛋白表达水平显著高于对照组rVV-Luc+(分别为P<0.05和P<0.01);CCK8结果表明,rVV-CCL5-SSTR2-Luc+对人胚肾上皮细胞HEK293T杀伤活力极低,而rVV-CCL5-SSTR2-Luc+对两株肿瘤细胞系的杀伤活力均随感染时间逐渐增强。并且rVV-CCL5-SSTR2-Luc+对肿瘤细胞的杀伤活力较之对照组rVV-Luc+均显著升高(HCT116:感染48 h时P<0.05;Hela:感染48 h时P<0.05,72 h时P<0.01)。结论本研究成功构建并获得了纯化的重组痘病毒rVV-CCL5-SSTR2-Luc+,并初步验证了其杀瘤活性,为开展后续体内外实验奠定了实验基础。

Objective The aim of this study is to construct a recombinant oncolytic vaccinia virus with CCL5 and SSTR2 genes and to detect its antitumor activity.Methods Recombinant vaccinia virus eukaryotic expression plasmid pSC65-CCL5-SSTR2-Luc+was constructed.The purified recombinant oncolytic vaccinia virus rVV-CCL5-SSTR2-Luc+was obtained after homologous recombination with WR vaccinia virus and 23 rounds of screen-ing and purification.The concentrated recombinant oncolytic vaccinia virus was abtained by expanded culture,concentration and purification.The expression changes of CCL5 and SSTR2 in HeLa cells after oncolytic vaccinia virus infection were determined by ELISA and Western blot respectively.The killing activity of rVV-CCL5-SSTR2-Luc+on two tumor cell lines was detected by CCK-8.Results The recombinant vaccinia eukaryotic expression plasmid pSC65-CCL5-SSTR2-Luc+was constructed successfully;Purified recombinant oncolytic vaccinia virus rVV-CCL5-SSTR2-Luc+with titer as 9.4×10^8 PFU/mL was obtained after homologous recombination,screening,purification and concentration;ELISA and Western blot results showed that after HeLa cells were infected with rVV-CCL5-SSTR2-Luc+,the expression level of CCL5 and SSTR2 protein in HeLa cells was significantly higher than those in the control group rVV-Luc+(P<0.05 and P<0.01,respectively);The results of CCK8 indicated that the killing activity of rVV-CCL5-SSTR2-Luc+on human embryonic renal epithelial cells HEK293T was very low,while the killing activity of rVV-CCL5-SSTR2-Luc+on two tumor cell lines increased gradually with the infection time.In addition,the killing activity of oncolytic vaccinia virus rVV-CCL5-SSTR2-Luc+on tumor cells was significantly increased as compared with that of the control group rVV-Luc+(HCT116:48 h after infection P<0.05;Hela:at 48 h,P<0.05;at 72 h,P<0.01).Conclusions Purified recombinant oncolytic vaccinia virus rVV-CCL5-SSTR2-Luc+is constructed and purified successfully,its tumoricidal activity is verified preliminarily,so this conclusion lays out an experimental foundation for subsequent in vivo and in vitro researches.