分子佐剂修饰的血管内皮生长因子融合蛋白疫苗抗肿瘤活性研究

Anti-tumor effects of vascular endothelial growth factor fusion protein vaccine modified by a molecular adjuvant

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 2 repeats of mycobacterial microbial hot shock protein 70（ HSP70） 407-426（ 2mHSP70407-426,M_2） could act as an effective molecular adjuvant to enhance the anti-tumor efficiency of vascular endothelial growth factor（ VEGF） fusion protein vaccine. Methods The gene encoding two tandem repeat sequences of HSP70407-426 was introduced to the terminus of the constructed hVEGF121 mutant 1 gene to prepare a recombined hVEGF121 mutant 2protein vaccine by PCR technique. The anti-tumor efficacy of hVEGF121 mutant 2 protein vaccine was investigated using a H22 liver cancer subcutaneous tumor model. The 24 mice were randomly divided into three groups： model group,experiment 1 group（ 80 μg hVEGF121 mutant 1 protein） and experiment 2 group（ 80 μg hVEGF121 mutant 2protein）. The humoral and cellular immune responses were detected by ELISA and splenic lymphocyte proliferation assay. The anti-angiogenesis effect was evaluated by an intradermal tumor model. Results Thetumor weight of the experimental 1 group and the experimental 2 group were（ 1. 17 ± 0. 25） g and（ 1. 58 ± 0. 28） g.Compared with the experimental 1 group,the tumor weight of tumor bearing mice in the experimental 2 group was lower significantly（ P〈0. 05）. The anti-VEGF antibodies of the experimental 1 group and experimental 2 group was 0. 54 ± 0. 09,0. 74 ± 0. 1. Compared with experimental 1 group,higher titers of anti-VEGF antibody were detected in immune serum of the experimental 2 group with statistically significant（ P〈0. 05）. The proliferative activity of spleen lymphocytes in the experimental 1 group and the experimental 2 group was 0. 26 ± 0. 03,0. 36 ± 0. 04. Compared with the experimental 1 group,the vaccine immunization in experimental 2 group can stimulate the proliferation of spleen lymphocytes more effectively with statistically significant（ P〈0. 05）. Conclusion M_2 could act as an effective adjuvant to help VEGF fusion protein vaccine to elicit a strong anti-tumor efficacy.