胰腺靶向的类表皮生长因子生物合成与靶向性研究

The Study of Pancreas-targeted Epidermal Growth Factor Biosynthesis and Targeting

目的为提高表皮生长因子(EGF)的稳定性和靶向性,利用大肠杆菌作为宿主细胞,设计和生物合成具有胰腺靶向的EGF融合蛋白。方法使用实验室已构建的含有胰腺靶向肽TRC(CHVLWSTRC)和重组聚多肽(Pastag200)修饰EGF基因的重组质粒,将重组质粒导入大肠杆菌进行EGF融合蛋白表达,采用亲和层析等技术纯化,WesternBlot鉴定。采用小鼠胚胎成纤维细胞(NIH/3T3)和胰岛内皮细胞(MS1)测试其生物学功能。使用KM小鼠探究EGF融合蛋白体内靶向效应。结果琼脂糖凝胶电泳和测序结果表明重组载体构建成功。WesternBlot技术验证EGF融合蛋白特异性。2种EGF融合蛋白对NIH/3T3细胞都具有一定的促增殖的能力(P<0.01),其中EGF-Pastag200-TRC表现出更高生物活性和结构稳定性,10 pg·mL^(-1) EGFPastag200-TRC对MS1细胞具有显著的促增殖效果。腹腔注射后,EGF-Pastag200-TRC在胰腺组织中的分布明显高于其他组织(P<0.01),证明其对胰腺组织具有靶向性。结论经具有胰腺靶向功能多肽与可增强稳定性聚多肽修饰的EGF显示出更高的生物学活性和稳定性,表现出良好的胰腺靶向性,为EGF用于糖尿病治疗提供了新思路。

OBJECTIVE To design and biosynthesis a pancreas-targeted epidermal growth factor(EGF)fusion proteinusing Escherichiacoli as a host cell to improve the stability and targeting ability of EGF.METHODS The recombinant plasmids containing the pancreatic targeting peptide TRC(CHVLWSTRC)and the recombinant polypeptide(Pastag 200)modified EGF gene were introduced into E.coli for EGF fusion protein expression,purified by affinity chromatography and identified by Western blot.The biological functions were verified using mouse embryonic fibroblasts(NIH/3T3)and pancreatic islet endothelial cells(MS1).The in vivo targeting effects of the EGF fusion protein were investigated using KM mouse.RESULTS The results of agarose gel electrophoresis and sequencing indicated that the recombinant vector was successfully constructed.Western blot technique verified the specificity of the ECF fusion protein.EGF-Pastag200-TRC fusion protein had certain pro-proliferative ability on NIH/3T3 cells(P<0.01)and showed higher biological activity and structural stability.Besides,10 pg·mL^(-1)EGF-Pastag200-TRC had a significant pro-proliferative effect on MS1 cells.After intraperitoneal injection,the distribution of EGF-Pastag200-TRC was significantly higher in pancreatic tissues than in other tissues(P<0.01),demonstrating its targeting ability to pancreatic tissues.CONCLUSION ECF modified with pancreatic targeting peptides and Pastag200 shows higher biological activity and enhanced stability.It also shows good pancreatic targeting ability in this study,and providesa new idea for the use of ECF in the treatment of diabetes.