融合蛋白CXCL10-loop3-EGF的可溶性表达及其抗肿瘤效应研究

Soluble expression of a CXCL10-loop3-EGF fusion protein and its anti-tumor activity

目的:构建重组趋化因子CXCL10及表皮生长因子受体干扰序列融合蛋白(CXCL10-loop3-EGF),验证其靶向性抗肿瘤效应及抑制血管形成活性。方法:运用RT-PCR从经IFN-γ刺激活化的PBMC中扩增人CXCL10基因,运用重叠延伸PCR法扩增CXCL10-loop3-EGF融合基因,并将其与载体pTG19-T连接、转化大肠杆菌DH5α、筛选阳性克隆,CXCL10-loop3-EGF融合基因与载体pET32a(+)连接、转化大肠杆菌Origami B(DE3),诱导可溶性表达重组his-CXCL10-loop3-EGF融合蛋白,并经镍柱亲和层析、酶切、超滤及透析等方法获得纯化的重组CXCL10-loop3-EGF融合蛋白。通过Transwell细胞趋化实验及HUVEC血管形成抑制实验验证此融合蛋白的抗肿瘤效应。结果:SDS-PAGE和Western blotting证实CXCL10-loop3-EGF融合蛋白构建成功,纯化后的融合蛋白具有显著的趋化活化PBMC活性及抑制HUVEC血管形成活性。结论:成功构建融合蛋白CXCL10-loop3-EGF,该蛋白具有较好的靶向性抗肿瘤活性。

AIM ： To evaluate the implication of CXCL10 - loop3 - EGF fusion protein for the activities of targeting tumor and anti - angiopoiesis. METHODS ： RT - PCR was preformed to amplify CXCL10 coding sequence from PBMC activated by IFN - γ CXCL10 - loop3 - EGF fusion gene, which was conducted by Over - Lap Extention PCR, was hinged up with plasmid pTG19 -T, transfected to E. coli DH5α and processed positive colony selection. After ligated with plasmid pET32a（ ＋ ）, recombinant CXCL10 - loop3 - EGF fusion gene was then transfected to E. coli Origami B （ DE3 ） and induced to express its coding fusion protein his - CXCL10 - loop3 - EGF. The recombinant fusion protein CXCL10 - loop3 -EGF was purified by His - bind affinity chromatograph, enterokinase cleavage, ultrafiltration and dislysis. The transwell chemotatic test and HUVEC angiopoiesis inhibition test were performed to determine the anti - tumor responses and antiangiopoiesis activity of CXCL10- loop3 -EGF fusion protein. RESULTS： CXCL10 -loop3 -EGF fusion protein was successfully constructed and confirmed by SDS - PAGE analysis and Western blotting. Significant PBMC chematatic activity and HUVEC anti - angiopoiesis activity were observed. CONCLUSION： CXCLIO - loop3 - EGF fusion protein. which has perfect anti -tumor activity, is successfully constructed.