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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
引用本文:陈敏方,陈利华,谢丽云,徐芬芬,夏爱晓,林忠. 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[J]. 中国临床药理学与治疗学, 2021, 0(1)
作者姓名:陈敏方  陈利华  谢丽云  徐芬芬  夏爱晓  林忠
作者单位:温岭市中医院胸外科;浙江省台州医院临床药学实验室;浙江省台州医院公共实验平台
基金项目:台州市科技基金(1802ky16);浙江省卫生厅科研基金(2015KYB436)。
摘    要: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。

关 键 词:VEGFR2  脂质体  pCMV质粒  疫苗

Preparation and the effect of antitumor of DNA plasmid lipidosome vaccine based VEGFR2 extracellular region by immunization activated in vitro
CHEN Minfang,CHEN Lihua,XIE Liyun,XU Fenfen,XIA Aixiao,LIN Zhong. Preparation and the effect of antitumor of DNA plasmid lipidosome vaccine based VEGFR2 extracellular region by immunization activated in vitro[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2021, 0(1)
Authors:CHEN Minfang  CHEN Lihua  XIE Liyun  XU Fenfen  XIA Aixiao  LIN Zhong
Affiliation:(Department of Thoracic Surgery,Wenling Traditional Chinese Medicine Hospital,Taizhou 317500,Zhejiang,China;Clinical Pharmacy Laboratory,Taizhou Hospital of Zhejiang Province,Taizhou 317000,Zhejiang,China;Public Experimental Platform,Taizhou Hospital of Zhejiang Province,Taizhou 317000,Zhejiang,China)
Abstract: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.
Keywords:VEGFR2  lipidosome  pCMV plasmid  vaccine
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