摘 要 目的: 血管内皮生长因子受体-2(vascular endothelial factor recepto-2,VEGFR-2)在肿瘤生长和转移过程中起着重要作用，制备口服型VEGFR-2 DNA疫苗并研究其生物免疫效应。方法：采用基因重组技术构建VEGFR-2胞外区基因表达载体pcDNA3.1-VR-2， DNA序列分析证实后，脂质体法转染COS-7细胞， Western blotting检测其VEGFR蛋白表达；以氯化钙法将pcDNA3.1-VR-2转化减毒沙门菌SL3261，制备口服型VEGFR-2 DNA疫苗。口服型疫苗免疫C57BL/6小鼠，ELISA法检测免疫后小鼠外周血VEGFR-2抗体水平，MTT法检测小鼠脾淋巴细胞对小鼠血管内皮细胞的体外杀伤活性。结果：成功制备口服型VEGFR-2 DNA疫苗。ELISA法检测显示，疫苗组小鼠抗体滴度随着免疫时间和免疫次数的增加而增高，在免疫6周后产生了高水平抗VEGFR-2 IgG类抗体(1.07±0.018)，明显高于生理盐水组(0.14±0.033)和质粒对照组(0.14±0.038)，差异均有统计学意义（F=853.17，P=0.000）。MTT法检测显示，疫苗组小鼠脾淋巴细胞对靶细胞MS1的杀伤率随着效靶比的增加而增加，在效靶比8∶1时CTL活性(89.38±1.51)明显高于生理盐水组(15.17±2.54)和空质粒对照组(12.99±4.31)，差异有统计学意义(F=315.42,P=0.000)。结论：成功制备了口服型VEGFR-2 DNA疫苗，该疫苗可激活小鼠特异性的细胞免疫和体液免疫，为进一步抗肿瘤研究奠定了基础。

Abstract Objective: Vascular endothelial growth factor receptor2 plays an important role in tumor growth and metastasis. This study is to prepare an oral VEGFR2 DNA vaccine and to evaluate its immune effects in mice. Methods: The recombinant plasmid pcDNA3.1VR2 targeting the extracellular domains of VEGFR2 was constructed by gene engineering technique. After confirmated by sequencing the pcDNA3.1VR2 vector was transfected into COS7 cells via lipid; the protein expression of VEGFR was identified by Western blotting. Then pcDNA3.1VR2 was transformed into attenuated Salmonella typhimurium SL3261 to develop an oral DNA vaccine against the extracellular domains of VEGFR2. C57BL/6 mice were immunized with the prepared oral vaccine; the serum level of VEGFR2Ag specific antibody was detected by ELISA. The cytotoxic T lymphocyte (CTL) was detected by modified MTT assay. Results：We successfully prepared VEGFR2 DNA vaccine. ELISA results showed that the VEGFR2specific IgG level in oral vaccine group (1.07±0018) was significantly higher than those of the empty vector group (0.14±0.038) and saline group (0.14±0.033)（F=853.17，P=0.000）. The titer of serum antibody increased with the immunization period and the immunization times. As shown by MTT assay, the prepared oral vaccine had specific and efficient cytotoxicity against murine endothelium cells （Ms1） in a dosedependent manner in vitro. The CTL activity in the oral vaccine group (89.38±1.51) was significantly higher than those of the empty vector group (12.99±4.31) and saline group (15.17±2.54) （F=315.42，P=0.000）．Conclusion：We have successfully prepared an oral VEGFR-2 DNA vaccine which can stimulate specific cellular and humoral immune responses in mice, paving a way for further anticancer research.

云南省科技厅昆明医学院联合专项基金资助项目(No. 2007C0024R)