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目的旨在获得p185胞外区及其亚区基因在原核系统中表达的多肽,并分析其与单抗结合特性。方法以pGEX-4T-1为载体,克隆p185胞外区及其亚区基因,并在原核系统中表达,经变性、复性后,以亲和层析法获得纯化的多肽。进一步通过ELISA方法分析了这些多肽与抗p185单抗A18的结合特性。结果研究获得原核系统表达的p185胞外区及其亚区多肽,GST-E3和GST-E3-4与单抗A18具有特异结合活性。结论原核系统表达的p185胞外区及其亚区多肽是可行的,初步确定为与单抗结合的亚区多肽,为进一步研究单抗识别的表位及其抑瘤机制奠定基础。
The purpose is to obtain the polypeptide expressed in the prokaryotic system of the p185 extracellular region and its sub-region genes, and analyze their binding characteristics with the monoclonal antibody. Methods pGEX-4T-1 was used as a vector to clone the extracellular region of p185 and its sub-region genes and expressed in prokaryotic system. After denaturation and renaturation, purified peptides were obtained by affinity chromatography. The binding properties of these polypeptides to anti-p185 mAb A18 were further analyzed by ELISA. Results The p185 extracellular region and its sub-region polypeptides expressed in prokaryotic system were obtained. GST-E3 and GST-E3-4 had specific binding activity with monoclonal antibody A18. Conclusion The p185 extracellular domain and its sub-region polypeptides expressed in prokaryotic system are feasible, and it is preliminarily identified as a subdomain polypeptide bound to mAb, which lays a foundation for further study on the epitope recognized by monoclonal antibody and its anti-tumor mechanism.