摘 要 目的: 构建cEGFR-rFc融合DNA疫苗， 观察其对小鼠黑素移植瘤的抑制作用。方法: 以异种同源鸡EGFR(chicken EGFR,cEGFR)膜外部分与兔疫球蛋白IgG的Fc段(rabbit IgG Fc,rFc)为基础构建真核表达载体pVAX1/cEGFR-rFc，脂质体法转染黑素瘤B16 细胞，免疫荧光法检测融合蛋白在B16细胞中的表达；以融合DNA疫苗免疫C57BL/6J小鼠，Western blotting法检测融合蛋白在小鼠体内的稳定表达。疫苗免疫小鼠接种B16黑素瘤细胞，14 d后处死部分小鼠，取出瘤体称重，计算抑瘤率；同时观察 小鼠荷瘤后的生存率；ELASIA法检测DNA疫苗免疫小鼠血清抗EGFR抗体滴度，流式细胞仪测定小鼠脾细胞淋巴细胞亚群。结果: 成功构建融合DNA疫苗pVAX1/ cEGFR-rFc，重组载体转染B16细胞后能检测到融合蛋白显著表达，疫苗免疫小鼠后能检测融合蛋白体内稳定表达。融合DNA疫苗能够延缓小鼠黑素移植瘤的生长，抑瘤率为54％(P<0.01)；能延长荷瘤小鼠的生存期(疫苗组小鼠接种瘤细胞后30 d的生存率为40%)；融合DNA疫苗诱发小鼠产生高滴度抗EGFR抗体(效价1∶1 000)和T细胞免疫(疫苗组小鼠脾脏CD8+T淋巴细胞数目显著增加, P<0.01)。结论: cEGFR-rFc融合DNA疫苗能产生有效对抗黑素瘤B16细胞的免疫效应。

Abstract Objective: To construct the recombinant DNA vaccine by fusing cEGFR with IgG Fc, and to observe the inhibitory effect of the constructed DNA vaccine against the transplanted melanoma in mice. Methods: The extracellular domain of xenogeneic chicken epidermal growth factor receptor (cEGFR) was fused with rabbit IgG Fc; and the product was insert into PVAX1 eukaryotic expression vector to construct the recombinant pVAX1/cEGFRrFc DNA vaccine. Expression of the fusion protein in melanoma B16 cells was detected by immunofluorescent assay after transfection. In vivo expression of the fusion protein in immunized C57BL/6J mice was detected by Western blotting assay. The DNA vaccines were used to immunize C57 mice 3 times before transplantation of B16 melanoma cells . The weights of tumors were determined after 14 d to calculate the antitumor rate and the survival rate of the mice was observed. The serum level of the specific antibody against human EGFR was determined by ELISA. Subgroups of spleen T lymphocytes were examined by FACS. Results: The eukaryotic expression vector pVAX1/ cEGFRrFc was successfully constructed. Effective expression of fusion protein was detectable in B16 cells after transfection with recombinant plasmid and stable expression was detected in mice after vaccine immunization. The fused DNA vaccine inhibited the growth of transplanted melanoma, with the inhibitory rate being 54% (P<0.01). The vaccine also prolonged the survival period of the mice transplanted with B16 cancer cells (the survival rate was 40% 30 d after inoculation). The vaccine also induced production of specific antibody against human EGFR (1∶1 000) and T cell immunoresponse (The CD8+ subgroups of spleen T lymphocyte were significantly increased \[P<0.01\]). Conclusions: The DNA vaccine containing cEGFR and IgG Fc is an effective antitumor vaccine against EGFRpositive tumor.

上海市重大科技攻关项目（No. 05DZ19309）