摘要
目的研究VEGF/bFGF复合多肽(VBP3)对人脐静脉血管内皮细胞(HUVEC)血管新生和人卵巢癌SKOV3细胞增殖抑制的影响。方法通过镍离子柱亲和层析和离子交换层析方法纯化出诱导表达的VBP3蛋白,免疫Balb/c小鼠制备抗bFGF和抗VEGF的多克隆抗体,酶联免疫吸附试验(ELISA)方法检测抗体效价和特异性。成管试验研究抗体对HUVEC细胞的成管影响,CCK-8检测抗体对SKOV3增殖抑制的影响。Western blot检测抗体对bFGF和VEGF下游信号通路中Erk1/2和Akt磷酸化的影响。结果 SDS-PAGE鉴定出了纯度较高的VBP3蛋白;ELISA方法检测出了高滴度的抗体;HUVEC的成管实验结果表明VBP3多克隆抗体在一定程度上抑制了HUVEC的成管;CCK-8检测出抗体体外抑制了SKOV3细胞增殖,在抗体质量浓度为100μg/ml时抑制率达到39.20%。Western blot结果表明抗bFGF和抗VEGF多克隆抗体显著抑制FGFR和VEGFR下游信号通路中Erk1/2和Akt的磷酸化。结论 VBP3可能作为一种多肽疫苗抑制肿瘤的生长。
This study designed to investigate the effects of antibodies from VEGF/bFGF complex peptide （VBP3） immunized mice on human umbilical vascular endothelial cell （HUVEC） angiogenesis and human ovarian cancer SKOV3 cells growth. VBP3 was expressed in E. coli with IPTG induction and purified with Ni-NTA affinity chromatography and anion exchange chromatography, then used to immunize Balb/c mice for preparing anti-VEGF and anti-bFGF antibodies. The antibody titer and specificity were analyzed by ELISA. CCK-8 method was used to investigate the function of these antibodies in inhibiting blood vessel formation of HUVEC cells and proliferation of SKOV3 ceils, while Western blotting was employed to detect the phosphorylation level of Erk1/2 and Akt. The SDS-PAGE results showed that VBP3 protein was expressed at the size of 30 KDa, and ELISA results showed that high titer anti-VEGF and anti- bFGF antibodies were prepared in Balb/c mice. Furthermore, the tube formation of HUVEC cells and proliferation of SKOV3 cells were significantly inhibited by these antibodies, and the SKOV3 cells inhibition rate was about 39.20% at 100 μg/ml of the antibodies. Also the antibodies resulted in the decrease of phosphorylation level of Erk1/2 and Akt assayed by the Western blot. The results revealed that the VBP3 may be used as a vaccine for tumor therapy in the future.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2014年第6期512-515,538,共5页
Immunological Journal
基金
国家自然科学基金(81171446)
中央高校基本科研业务费专项资金(21612112)
