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设计并构建一种含VEGF和GRP抗原表位的表达载体p ET28a-VEGFI-M2-GRP质粒,将其转化至大肠杆菌中,并对重组菌进行乳糖诱导表达,超声破碎,包涵体经洗涤、溶解、透析复性、离子交换层析等方法进行分离纯化后得到目的蛋白VEGFⅡ/GRP,Western blot鉴定。建立前列腺癌RM-1细胞的C57BL/6J小鼠皮下移植瘤模型,以VEGFⅡ/GRP作为疫苗进行免疫。观察荷瘤小鼠的肿瘤生长情况计算抑瘤率,比较各组血管生成数以及ELISA检测抗VEGF抗体浓度,并研究该蛋白疫苗的抗肿瘤生长作用和抗血管生成作用。结果显示:ELISA结果表明小鼠血清中抗VEGF抗体比NS组高(P<0.05),重组蛋白VG组与NS组相比抗血管作用显著(P<0.05)。初步显示构建的重组蛋白有抑制肿瘤血管生长的作用。
The plasmid p ET28a-VEGFI-M2-GRP containing the VEGF and GRP epitopes was designed and constructed and transformed into Escherichia coli. The recombinants were induced by lactose, sonicated and washed. Lytic, refolding, ion exchange chromatography and other methods were isolated and purified to obtain the target protein VEGF Ⅱ / GRP, Western blot identification. A subcutaneous xenograft model of C57BL / 6J mice bearing prostate cancer RM-1 cells was established and immunized with VEGFII / GRP as a vaccine. The growth inhibition rate of tumor-bearing mice was observed to calculate the tumor inhibition rate. The number of angiogenesis in each group and the concentration of anti-VEGF antibody in each group were compared. The antitumor activity and anti-angiogenic effect of the vaccine were also studied. The results showed that the anti-VEGF antibody in serum of mice was higher than that of NS (P <0.05). The anti-VEGF of recombinant protein VG was significantly higher than that of NS (P <0.05). Preliminary results show that the constructed recombinant protein can inhibit tumor angiogenesis.