脂质体VEGFR2胞外区基因肿瘤疫苗的制备及其免疫激活体外抗肿瘤活性研究

目的:制备脂质体(LP)携带VEGFR2胞外区(exVEGFR2)肿瘤抗原pCMV质粒复合物基因疫苗,为肿瘤免疫治疗提供新的主动免疫疗法。方法:RT-PCR扩增含2个KpnⅠ和XbaⅠ限制性酶切位点的exVEGFR2序列,将其与pCMV质粒连接,构建pCMV/exVEGFR2质粒。Western blot检测exVEGFR2体外表达水平。用制备好的脂质体pCMV/exVEGFR2复合物(LP-pCMV/exVEGFR2)免疫C57BL/6小鼠,ELISA法检测其免疫激活活性。利用51 Cr释放实验分析CTLs介导的细胞毒活性。结果:成功扩增到exVEGFR2序列,并构建pCMV/exVEGFR2质粒;只有在pCMV/exVEGFR2转染COS-7细胞中,检测到与目的蛋白序列大小一致的相对分子质量为90000的VEGFR2特异性条带。LP-pCMV/exVEGFR2免疫小鼠6周后,ELISA法检测到强烈的特异性抗VEGFR2免疫激活效应;其免疫T细胞能够有效地介导体外VEGFR2阳性的CT-26结肠癌细胞毒性。结论:LP-pCMV/exVEGFR2能够有效地激活小鼠产生特异性抗VEGFR2免疫应答效应,并产生体外抗肿瘤细胞免疫毒性,为后期研究主动免疫治疗VEGFR2阳性肿瘤奠定实验基础。

Preparation and the effect of antitumor of DNA plasmid lipidosome vaccine based VEGFR2 extracellular region by immunization activated in vitro

AIM:The DNA plasmid lipidosome(LP)vaccine based VEGFR2 extracellular region(exVEGFR2)was prepared in order to provide a new approach for cancer active immunotherapy.METHODS:High fidelity PCR was used to amplify the target sequence of exVEGFR2 with two restriction site of KpnⅠand XbaⅠ.The plasmid of pCMV/exVEGFR2 was constructed by connected exVEGFR2 with pCMV empty plasmid.The activity of immune activation was detected by ELISA.CTLs mediated cytotoxic activity was analyzed by 51 Cr release assay.RESULTS:The 90000 target specific band of VEGFR2 was detected by Western blot.After 6 weeks since the first time vaccination,an intense specific immune response of anti-VEGFR2 was detected by ELISA in the serum from the C57BL/6 mouse vaccinated with LP-pCMV/exVEGFR2 vaccine.The T cells from the spleen of mouse immunized with the vaccine induced the cytotoxicity effect on CT-26 with VEGFR2 positive.CONCLUSION:The results of the specific immune response of anti-VEGFR2 in vivo and the antitumor cytotoxicity in vitro by vaccinated with LP-pCMV/exVEGFR2 vaccine in mouse model,would lay the foundation for further study of VEGFR2 positive tumor treated in vivo by the active immunotherapy.