人表皮生长因子原核表达及活性鉴定

Human epidermal growth factor expressed in prokaryotic cells and function identification

人表皮生长因子(hEGF)是由53氨基酸组成的小分子多肽,它通过与膜受体的相互作用调节多种类型的细胞增殖。为获得具有生物活性的基因工程产品hEGF,设计在hEGF基因5’-端插入功能序列与基因融合,经合成基因、双酶切连接,构建了重组原核表达载体pBV-F-hEGF,并导入大肠杆菌HB101菌株。工程菌在37℃LB培养基中培养至对数生长期,并在42℃温度条件下诱导表达蛋白F-hEGF。以5000 r/min离心收集细胞,利用超声波破碎细胞,以10000 r/min离心20 min收集包涵体沉淀。含有F-hEGF的包涵体溶解后,采用Ni-NTA柱纯化目的蛋白,并用SDS-PAGE鉴定目的蛋白。利用免疫组化反应和MTT方法分析F-hEGF与其膜受体EGFR的特异性结合活性和细胞增殖活性。结果显示,融合蛋白F-hEGF得到高效表达,表达量在35%左右,纯化的蛋白浓度为3.1 mg/mL,F-hEGF与膜受体能发生特异性结合,具有明显的促细胞增殖活性。本研究构建了表达生物活性F-hEGF的工程菌,为hEGF进一步的临床和开发应用提供依据。

Human epidermal growth factor(hEGF)is a small polypeptide composed of 53 amino acids,which mediates many types of cell proliferation through interaction with its membrane receptor.In order to obtain the bioactive hEGF,a fusion hEGF(F-hEGF)gene with a functional sequence on 5’-end of hEGF gene was designed.After the gene was synthesized,digested by double restriction enzyme and ligated,recombinant prokaryotic expression vector pVB-F-hEGF was constructed and transformed into Escherichia coli strain HB101.The engineering strain was cultured in LB culture at 37℃until logarithmic growth phase and F-hEGF was induced to express in E coli cells under 42℃temperature condition.Expression cells were centrifuged and collected at 5000 r/min,smashed into pieces with ultrasonic wave.The broken cell solution was centrifuged at 10000 r/min for 20 mins and the inclusion body precipitation was collected.After the inclusion body containing F-hEGF was dissolved,the desired protein was purified using Ni-NTA column and identified with SDS-PAGE electrophoresis.The specific interaction between F-hEGF and EGF receptor(EGFR)on target cell membrane and F-hEGF promoting proliferation were detected by immunohistochemical reaction and MTT experiment.The results show that F-hEGF protein is effectively expressed at circa 35%and 3.1 mg/mL,when it could specifically interact with its membrane receptor and obviously enhanced cell proliferation.This study constructs the engineering strain expressed bioactive F-hEGF and lays the groundwork for hEGF further development and application in clinic and cosmetic medicine.