Jichu yixue yu linchuang (Apr 2020)
Construction of three strains of recombinant oncolytic vaccinia virus and their anti-tumor activity
Abstract
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 9.4×108 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.
