| HER2is closely related to the occurrence, development and prognosis of breastcancer, and is an important prognosis factor in breast cancer. HER2is overexpressedin tumor tissues, mediates cancer formation, and locateded at the cell surface, whichmake it an ideal anti-tumor therapeutic targets. Untill now, most HER2antibodiesacquired took the entire extracellular region of HER2as antigens. While in this paper,the particular parts of extracellular functional domains were chosen as targeting pointsbased on the small molecule advantage of VHH nanobodies. Thus we can obtainspecific anti-HER2VHH nanobodies by phage display screening. And they wereexpectated to be applied in clinical diagnosis and treatment.The fragment of HER2extra-cellular domain (ECD) cDNA was obtained by RT-PCR from human breast cancer cell line MCF-7, and cloned into the pGEX-4T-1vector．Then HER2-II and HER2-IV gene fragments were amplificated by PCR andused to construct recombinant expression vectors pGEX-4T-1-HER2-II and pGEX-4T-1-HER2-IV. GST-HER2-II and GST-HER2-IV fusion protein could be highlyexpressed in E. coli BL21by optimizing inducing conditions, which was to induce theinoculums (OD600=0.6) at20-28℃, in the presence of0.1mM IPTG for about9-16h.Then collected the bacaterial cells and had it ultrasonic sonicated. The supernatantobtained was purified by affinity chromatography, and then was desalted andconcentrated to obtain the recombinant antigens, whose purity and concentration wastested later. Coated the recombinant antigens at solid phase and then added in VHHnanobody library. After three rounds of selection processes of “adsorption-elution-amplification”, ELISA was used to identify specific VHH nanobodies which hadaffinity with targeted antigens from96randomly selected clones, and finally one wasidentified. This subject is expected to ultimately provide for the clinical treatment anew high titer, low side effects, low cost, small-molecule antibody drugs. |
PDF Full Text Request