双歧杆菌表达的重组人GrB-VRB融合蛋白诱导KDR阳性细胞凋亡

Granzyme B-VEGF receptor-binding peptide fusion protein expressed in B.longum induces apoptosis of KDR-positive cells

目的在双歧杆菌中表达重组人颗粒酶B-血管内皮生长因子结合肽(GrB-VRB)融合蛋白并研究其对血管内皮生长因子受体I(IKDR)阳性细胞的作用。方法采用电穿孔法将重组表达载体转入双歧杆菌,以ELISA和免疫印迹鉴定表达产物,以细胞增殖试验和TUNEL法分析其对KDR阳性细胞的影响。结果工程化化双歧杆菌培养物的上清和菌体中均有重组产物,重组人GrB-VRB能有效抑制KDR阳性如人脐静脉血管内皮细胞株HUVEC、小鼠结肠癌细胞株CT-26细胞增殖、细胞死亡,这种死亡的机制是细胞凋亡;但对KDR阴性细胞则无这些作用。结论工程化双歧杆菌能分泌性表达重组GrB-VRB融合蛋白,其在VRB的靶向作用下,GrB可有效地诱导KDR阳性细胞凋亡。

Objective To express granzyme B-vascular endothelial growth factor （VEGF） receptor-binding peptide （GrB-VRB） fusion protein in Bifidobacteria longum （B. longum） and investigate the effects of this fusion protein on the proliferation and apoptosis of cells expressing VEGF receptor Ⅱ, the kinase domain receptor （KDR）. Methods The recombinant expression vectors pBBADx-VRB, pBBADx-GrB and pBBADx-GrB-VRB were separately transformed into B. longum cells by electroporation. The expressed products were identified by enzyme-linked immunosorbent assay and Western blotting, and their effects on KDR-positive cells were analyzed using proliferation assay and TUNEL assay. Results The expressed products were detected in both the supernatant and cellular fractions of B. longum cells. The recombinant GrB-VRB fusion protein reacted with such KDR-positive ceils as human umbilical vein endothelial cells （HUVEC） and mouse colon cancer cell line CT-26, and caused obvious cell proliferation inhibition, cytotoxicity and cell apoptosis in these cells. Conculsion The recombinant GrB-VRB fusion protein secreted by the engineered B. longum ceils can induce KDR-positive cell death as the result of GrB-induced cell apoptosis following the celI recognition by VRB.