Cloning And Expression In Escherichia Coli Of HER2Extracellular Domain

Objective: Human epidermal growth factor receptor2(HER2) oncogene is amember of the epidermal growth factor receptor (EGFR) family with intrinsicallytyrosine kinase activity. HER2consists of four domains: signal peptide, extracellulardomain (ECD), transmembrane domain and intracellular domain. HER2is seldomexpressed in normal cells, but is over expressed in cells and organizations of breastcancer, gastric cancer, ovarian cancer and gastric cancer. HER2extracellular domainlocated on the tumor cell surface, is regarded as the ideal target of tumorimmunotherapy. The purpose of this study is to build recombinant plasmid forexpression of HER2extracellular protein which is subsequently used for preparation ofspecific antibody or tumor protein vaccine.Methods:(1) Cloning of HER2extracellular domain template: The wholeextracellular domain gene of HER2was amplified by RT-PCR from human breastcancer cell line SKBR-3.(2) Construction of HER2protein expression vector: Thegenes of membrane-distal region and juxtamembrane region of HER2extracellulardomain were amplified from the cloned template, and then inserted into the expressionvector pET28a and pET30a respectively. The recombined vectors were sequenced usingT7universal primers.(3) Induced expression of target protein: The recombinantexpression vectors were transformed into E. coli BL21(DE3) cells and induced byIPTG at37℃, and the expressed proteins were detected by SDS-PAGE andWestern-blotting.(4) Inducing condition optimization: The optimal inducing conditionfor high expression of target proteins was determined by changing temperature andincubating time.Result:(1) Agarose gel electrophoresis showed a specific band near2000bpdemonstrating the successful cloning of HER2extracellular domain template.(2) DNAsequencing indicated the inserted DNA fragments completely accorded with the targetfragments, the plasmid construction succeed.(3) SDS-PAGE of the transformed bacteria showed specific bands corresponding with the target proteins. This indicatedthe target proteins were successfully expressed in E.coli as inclusion body.(4)Bacteria induced with IPTG at37℃for4h was the optimal condition for highexpression of target proteins.Conclusion: Two HER2extracellular domain proteins were successfully expressedby transforming E.coli with prepared recombinant plasmids. It lays the foundation forproduction of HER2specific antibody and study of tumor protein vaccine.