摘要
目的构建表达白细胞介素-18的条件增殖腺病毒ZD-IL-18并研究其介导IL-18对恶性黑色素瘤A375细胞的抗肿瘤活性。
方法以质粒pJW-IL-18为模板,PCR扩增获得编码成熟IL-18的基因序列。将IL-18基因克隆至pCA13质粒,构建重组质粒pCA13-IL-18。用限制性内切酶BglⅡ从pCA13-IL-18酶切出包含CMV启动子及IL-18基因的表达框,并克隆至条件增殖腺病毒质粒pZD55,构建重组质粒pZD55-IL-18。将pZD55-IL-18与含有腺病毒右臂的质粒pBHGE3共转染HEK293细胞,9~12天后出现病毒空斑。提取重组腺病毒的DNA,经PCR鉴定正确者即为条件增殖腺病毒ZD55-IL-18。重组腺病毒大量扩增,氯化铯梯度离心纯化,测定病毒滴度。ZD55-IL-18转染人恶性黑色素瘤A375细胞,通过MTT法检测肿瘤细胞的增殖抑制及结晶紫染色法分析细胞病变作用,逆转录-聚合酶链反应(RT-PCR)检测IL-18 mRNA的表达及免疫印迹法(Western Blotting)分析E1A与IL-18的表达。
结果成功构建了表达IL-18的条件增殖腺病毒ZD55-IL-18;经PCR鉴定表明ZD55-IL-18包含目的基因;ZD55-IL-18的滴度可达1×1011PFU/ml;Western Blotting的结果显示ZD55-IL-18在A375肿瘤细胞中表达E1A与IL-18;ZD55-IL-18抑制恶性黑色素瘤细胞株A375生长的作用显著优于Ad-IL-18。
结论条件增殖腺病毒ZD55-IL-18在恶性黑色素瘤细胞株A375中选择性增殖并高效表达IL-18,从而抑制肿瘤细胞A375的生长。该研究为进一步探索条件增殖腺病毒介导IL-18靶向治疗肿瘤提供了实验研究依据。
Objective To construct conditionally replicative adenovirus (CRAd) expressing Interleukin(IL)-18. To investigate the antitumor activity of replication competent adenovirus expressing IL-18 on malignant melanoma A375 cells in vitro.
Methods One pair of specific primers was designed and used to get human IL-18 cDNA sequences and change its restriction enzyme sites of BglⅡby polymerase chain reaction using pJW-IL-18 as the template. The PCR product was inserted into pCA13 plasmid to construct the recombinant plasmid (pCA13-IL-18). The pCA13-IL-18 plasmid was cut with BglⅡto get the expression cassette of IL-18 gene and then the expression cassette was cloned into pZD55 which has been cut with BglⅡand dephosphated to form pZD55-IL-18 plasmid. It was identified that the pZD55-IL-18 had been constructed successfully. The plasmid pZD55-IL-18 was transfected in HEK293 cells together with plasmid pBHGE3 to obtain the recombined CRAd, ZD55-IL-18. The viral plaques appeared 9~12 days after infection. The recombined adenoviruses, extracted DNA, were verified by PCR. Viruses were plaque purified, propagated on HEK293 cells and purified by CsCl gradient according to standard techniques, and functional PFU titers were determined by plaque assay on HEK293 cells. The antitumor potential of ZD55-IL-18 to A375 cells was evaluated by MTT assay and crystal violet dye method. The effect of ZD55-IL-18 on the IL-18 expression in mRNA levels of A375 cells was detected by RT-PCR. The expression of IL-18 and E1A protein was also detected by Western-blotting.
Results CRAd expressing IL-18 had been constructed successfully. The analysis of PCR indicated the recombinant adenovirus ZD55-IL-18 containing IL-18 gene and without wild adenovirus. Functional PFU titers of ZD55-IL-18 were 1×1011PFU/ml. Western-blotting analyses indicated ZD55-IL-18 can express E1A and IL-18 in adenovirus infected A375 cells. ZD55-IL-18 is more effective to inhibit the proliferation of A375 cells than Ad-IL-18 in vitro.
Conclusion CRAd ZD55-IL-18 can enhance the expression of IL-18 and inhibit the proliferation of malignant melanoma A375 cells. CRAd expressing IL-18 may be used for further investigation of Gene-Viro therapy of cancer.
引文
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