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目的:利用原核表达系统,构建异源血管内皮生长因子融合蛋白疫苗,研究该融合蛋白疫苗对小鼠Lewis肺癌肿瘤模型的抗血管生成主动免疫治疗效果。方法:将鸡VEGF基因克隆入pGEX-4T-2原核表达载体,经IPTG诱导融合蛋白表达,Ni-NTA纯化,透析复性,构建GST-cVEGF融合蛋白疫苗。将C57BL16J小鼠随机分为3组,GST-cVEGF疫苗组10只、cVEGF疫苗组10只、生理盐水组(NS)10只。将各组样品与等体积弗氏佐剂混合,各组小鼠每只分别0、2、3周免疫100μgGST-cVEGF融合蛋白疫苗、单纯cVEGF蛋白疫苗、生理盐水。接种Lewis肺癌细胞,观察肿瘤的生长情况和小鼠状态。接种肿瘤18d后,处死小鼠,剥离肿瘤称重。检测小鼠血清中特异性抗VEGF抗体滴度。结果:GST-cVEGF疫苗组、cVEGF组、生理盐水组肿瘤均重分别为(1·55±0·534)g、(1·93±0·607)g、(2·87±0·642)g。GST-cVEGF疫苗组肿瘤重量明显小于生理盐水组(P<0·01)。疫苗组血清产生抗VEGF抗体滴度为1∶2000。结论:异种血管内皮生长因子基因重组融合蛋白疫苗可干扰机体对自身VEGF的免疫耐受,可产生较高水平的特异性抗VEGF抗体,并具有抑制小鼠Lewis肿瘤生长的效应。
OBJECTIVE: To construct a prokaryotic expression vector of fusion protein vaccine by prokaryotic expression system and investigate the anti-angiogenic effect of the fusion protein vaccine against Lewis lung cancer in mice. Methods: The VEGF gene of chicken was cloned into prokaryotic expression vector pGEX-4T-2, and the fusion protein was induced by IPTG. Ni-NTA purification and dialysis refolding were performed to construct GST-cVEGF fusion protein vaccine. The C57BL16J mice were randomly divided into three groups: 10 in the GST-cVEGF vaccine group, 10 in the cVEGF vaccine group and 10 in the NS group. Each group of mice was mixed with an equal volume of Freund’s adjuvant. Each group of mice were immunized with 100 μg of GST-cVEGF fusion protein vaccine, cVEGF protein vaccine and normal saline at 0, 2 and 3 weeks respectively. Lewis lung cancer cells were inoculated and the growth of the tumor and the state of the mouse were observed. Twenty-eight days after inoculation, the mice were sacrificed and the tumors were dissected and weighed. Titers of specific anti-VEGF antibodies in sera of mice were measured. Results: The tumor weight in the GST-cVEGF vaccine group, the cVEGF group and the saline group were (1.55 ± 0.543) g, (1.93 ± 0.607) g, (2.87 ± 0.642) g. The tumor weight of GST-cVEGF vaccine group was significantly lower than that of saline group (P <0.01). The serum level of anti-VEGF antibody produced by the vaccine group was 1: 2000. CONCLUSION: The recombinant VEGF fusion protein vaccine can interfere with the immune tolerance of the human body to its own VEGF, produce high level of specific anti-VEGF antibody and inhibit the growth of Lewis tumors in mice.