EGF-linker-PE40重组毒素的构建、表达及其意义

Prokaryotic expression of EGF-linker-PE40 gene and its application

目的构建人表皮生长因子(EGF)-柔性连接肽(linker)-铜绿假单胞菌外毒素活性片段(PE40)重组毒素的表达载体并诱导表达,以探索其成为治疗肿瘤新型靶向药物的可能性。方法用PCR技术扩增出EGF和PE40基因,通过引物设计在两者之间加上19个中性氨基酸短肽(TS-(G4S)3-AC)的linker编码序列、克隆PCR产物,并构建pET22b(+)-EGF-linker-PE40表达质粒,将其转化E.coli BL21(DE3)后,诱导表达,SDS-PAGE及凝胶薄层扫描分析EGF-linker-PE40重组毒素的表达情况。结果 EGF-linker-PE40重组毒素在BL21(DE3)中可溶性表达,目的蛋白占细胞总蛋白的18%左右。结论应用基因工程技术,成功地构建了重组毒素EGF-linker-PE40的表达载体,并得到高效可溶性表达,为进一步研究其结构功能关系和肿瘤临床应用奠定了基础。

Objective To construct a prokaryotic plasmid pET22b（＋）-EGF-linker-PE40 for the expression of human epithelial growth factor（EGF）-flexible peptide（linker）-functional segment of Pseudomonas aeruginosa exotoxin peptide（PE40） and utilize the recombinant peptid as a molecular agent in cancer targed therapeutics.Methods EGF gene and PE40 gene were amplified by its specific primers respectively and linked by a polypeptide of 19 neutral amino acids（TS-（G4S）3-AC）.The recombinant peptid was cloned into pET22（＋） and then transformed into E.coli BL21.Induced by IPTG,the recombinat protein was analyzed by SDS-PAGE.Results The recombinant peptide was soluble expression and amounted to 18% of bactetia total protein.Conclusions EGF-linker-PE40 is highly expressed in the soluble form.This work lays the base for further study of its structure,function and application in cancer targed therapeutics.