OX-M-AdmCLB1重组腺病毒制备及其诱导的体内抗肿瘤效应

Construction of Oxidative Mannan-conjugated Adenovirus-mcyclin B1 and Induction of Anti-tumor Effect in Vivo

构建小鼠细胞周期蛋白B1(mcyclinB1)重组腺病毒(AdmCLB1),将氧化型甘露聚糖(OX-M)与AdmCLB1偶联,制备OX-M-AdmCLB1,研究OX-M-AdmCLB1诱导的抗肿瘤效应。利用AdEasy系统构建携带靶基因小鼠细胞周期蛋白B1的复制缺陷型重组腺病毒AdmCLB1,抽提AdmCLB1基因组DNA,进行PCR扩增。重组病毒经扩增、纯化后与OX-M混合,制备OX-M-AdmCLB1;OX-M-AdmCLB1体外感染小鼠树突状细胞(DC),RT-PCR扩增分析靶基因表达;OX-M-AdmCLB1处理BALB/C小鼠,处理后再荷瘤,观察小鼠肿瘤生长及生存情况。重组病毒AdmCLB1终滴度为2.1×1011pfu/ml;DC内靶基因的表达量在OX-M-AdmCLB1组较AdmCLB1组高;体内研究提示OX-M-AdmCLB1可明显抑制CT26肿瘤增殖(抑瘤率44%)、延长动物生存期(P<0.01)。实验制备的新型重组腺病毒OX-M-AdmCLB1体内能够诱导明显的抗肿瘤效应,估计此效应的产生与DC特异识别OX-M-AdmCLB1,进而激活机体抗肿瘤免疫有关。

To construct the replication-deficient recombinant adenoviruses inserted mouse cyclin B1 （mcyclin B1） cDNA drived by CMV promoter using homologous recombination in bacteria provided by AdEasy system. Then to synthesize oxidative mannan-conjugated adenovirus-mcyclin B1 （OX-M-AdmCLB1） and further elucidate its anti-tumor activity. The shuttle plasmid pShuttle-CMV-mcyclin B1 （pSh-C-mCLB1） in which mcyclin B1 cDNA was inserted into the downstream of CMV promoter was established by ligation. Then the linearized pShC-mCLB1 was co-transformed with backbone vector pAdEasy-1 to obtain the recombinant adenoviral plasmids pAdmCLB1 by homologous recombination. After packed in HEK-293 cells, the recombinant adenovirus AdmCLB1 was obtained. To further confirm AdmCLB1, its genomic DNA was isolated and used as template to gain mcyclin B1 cDNA by PCR amplification. AdmCLB1 was expanded and purified. To synthesize OX-M-AdmCLB1, OX-M was mixed with AdmCLB1. Dendritic cells （DCs） were infected with OX-M-AdmCLB1 in vitro and the expression of mcyclin B1 in DCs was evaluated through RT-PCR amplification. Being treated with OX-M-AdmCLB1 one week later, BALB/C mice were challenged with CT26 colon carcinoma （CT26） cells. Then the tumor growth and survival of mice were observed. The virus titer of AdmCLB1 was 2.1× 10^11 pfu/ml. The expression of mcyclin B1 in DCs infected with OX-M-AdmCLB1 was higher than AdmCLB1 group. The potential for attenuating tumor growth and sustaining survival benefits in mice treated with OX-M-AdmCLB1 has been displayed. OX-M-AdmCLB1 we constructed could induce the anti-tumor activity in vivo successfully, which might be connected tightly with the activation of immune system through the target recognition between DCs and OX-M-AdmCLB1.