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目的研究两段重复的热休克蛋白70(mHSP70)407-426序列(2mHSP70_(407-426),M_2)能否增强血管内皮生长因子(VEGF)融合蛋白疫苗抗肿瘤活性。方法以加端PCR方式,将M_2以柔性肽连接于hVEGF121突变体1的C末端,得到hVEGF121突变体2蛋白。用H22肝癌细胞构建荷瘤肝癌小鼠模型。24只小鼠按照体重随机分为3组:模型组,实验1组(hVEGF121突变体1蛋白80μg)及实验2组(hVEGF121突变体2蛋白80μg)。以酶联免疫吸附法检测免疫小鼠血清中的抗体水平,以脾淋巴细胞增殖实验检测免疫小鼠细胞免疫应答水平,以皮内肿瘤血管模型考察蛋白疫苗抗血管生成活性。结果实验1组与实验2组的瘤重分别为(1.58±0.28),(1.17±0.25)g,与实验1组比较,实验2组差异有统计学意义(P<0.05)。实验1组与实验2组的小鼠免疫血清中抗-VEGF抗体分别为0.54±0.09,0.74±0.1,与实验1组比较,实验2组小鼠免疫血清中检测到更高滴度的抗-VEGF抗体,差异有统计学意义(P<0.01)。实验1组与实验2组的刺激脾淋巴细胞的增殖活性分别为0.26±0.03,0.36±0.04,与实验1组比较,实验2组疫苗免疫能够更有效地刺激脾淋巴细胞的增殖,差异有统计学意义(P<0.01)。结论 M_2作为分子佐剂可以有效增强VEGF融合蛋白疫苗的抗肿瘤活性。
Objective To investigate whether the two repeated segments of 407-426 heat shock protein (mHSP70) 407-426 (2mHSP70_ (407-426) and M_2) can enhance the antitumor activity of VEGF fusion protein vaccine. Methods The hVEGF121 mutant 2 protein was obtained by indirect PCR with M_2 as a flexible peptide linked to the C terminus of hVEGF121 mutant 1. H22 hepatoma cells were used to construct mouse model of hepatocellular carcinoma. Twenty-four mice were randomly divided into three groups according to body weight: model group, experiment group 1 (hVEGF121 mutant 1 protein 80μg) and experiment group 2 (hVEGF121 mutant 2 protein 80μg). The level of antibody in sera of immunized mice was detected by enzyme-linked immunosorbent assay (ELISA). The level of cellular immune response in immunized mice was detected by splenic lymphocyte proliferation assay. The anti-angiogenic activity of the protein vaccine was examined by intradermal tumor vascular model. Results The tumor weights in experimental group 1 and experimental group 2 were (1.58 ± 0.28) and (1.17 ± 0.25) g, respectively. Compared with experimental group 1, the tumor weight in experimental group 2 was statistically significant (P <0.05). Compared with the experimental group 1, the anti-VEGF antibody in the mouse serum of experimental group 1 and experimental group 2 was 0.54 ± 0.09 and 0.74 ± 0.1, respectively. Compared with the experimental group 1, VEGF antibody, the difference was statistically significant (P <0.01). The proliferation activity of splenic lymphocytes in experimental group 1 and experimental group 2 were 0.26 ± 0.03 and 0.36 ± 0.04, respectively. Compared with experimental group 1, the immunization of experimental group 2 could more effectively stimulate the proliferation of splenic lymphocytes with statistical difference Significance (P <0.01). Conclusion M_2 as a molecular adjuvant can effectively enhance the anti-tumor activity of VEGF fusion protein vaccine.