防御素2融合VE-Cadherin重组质粒的抗血管生成免疫作用

The Antiangiogenic Effect of DNA Vaccine Based on β-defensin 2 Fused with VE-Cadherin

目的构建β防御素2(BD2)融合血管内皮钙粘附素(VE-Cadherin)胞外段重组质粒,以期探讨其免疫后抗肿瘤血管生成作用。方法PCR扩增成熟BD2片段和VE-Cadherin胞外段。以连接肽连接成熟BD2片段和VE-Cadherin胞外段,通过重叠PCR构建融合分子。将所得三种片段插入到pSecTag2B真核表达质粒。转染CT26肿瘤细胞,RT-PCR检测其表达。取转染COS细胞后的上清液做趋化实验,检测融合分子对树突状细胞(DC)的趋化作用。所得质粒经肌肉注射免疫小鼠后,皮下植入包裹CT26肿瘤细胞的藻酸盐微球,检测血管生成情况。结果测序证实三种质粒构建正确,能在真核细胞中顺利表达。BD2融合重组质粒和未融合的BD2质粒转染细胞后的上清液趋化DC的能力相近(P>0.05)。与其余各组质粒免疫相比,融合重组质粒免疫后能够显著抑制藻酸盐微球中肿瘤细胞诱导的血管生成(P<0.01)。结论BD2融合血管生成相关分子后的重组质粒免疫动物后能在体内有效地打破机体对肿瘤新生血管的免疫耐受。

Objective To explore the feasibility of generating beta defensin 2 fusion vaccine to break the immune tolerance of self antigens associated with angiogenesis. Methods PCR amplification of mature beta defensin 2 （Def） and extracelluar segment of Vascular Endothelial Cadherin （Cad） was conducted. （GGGS）3 was used as linker peptide for fusion plasmid（Def-Cad）. All the three segments were inserted into pSecTag2B plasmid （pSec）, and CT26 tumor cells were transfected. Expression was verified by RT-PCR and chemotaxis assay. The alginate bead model was used to study the antiangiogenic effect of fusion plasmid vaccination. Results All constructions were verified by sequencing and were found expressed in mammalian cell. The beta defensin 2 fusion protein had similar capacity to chemoattract dendritic cells as compared with nonfusion protein （P〉0. 05）. The alginate bead assay revealed that the tumor-induced angiogenesis in fusion plasmid immunized mice was significantly suppressed when compared with that in nonfusion plasmid （P 〈 0. 01）. Conclusion An active immunization strategy based on beta defensin 2 fused with angiogenesis related antigen of endothelial can inhibit angiogenesis and may be a useful approach for cancer therapy.