Design Of Fusion Protein Of Single Chain Antibody(SCFV)and Antitumor Polypeptide(ACP)and Its Antitumor Activity

Epidermal growth factor receptor(EGFR)is widely present in most solid tumors.Because its overexpression is closely related to the growth and metastasis of solid tumors,it is called an essential biomarker for cancer.Cetuximab is the most widely used EGFR antibody,and it is also used in the treatment of colorectal cancer,head and neck cancer and other cancers related to EGFR overexpression,which has greatly affected its treatment efficiency of clinical application due to the drawback of large size.The significant advantage of humanized single-chain antibody(huscFv)is that it has a small molecular weight and strong penetration.Because of its humanization,it can also eliminate part of the immunogenicity of mouse antibodies.This subject is based on the EGFR antibody cetuximab,and the VH and VL fragments are selected as the main structure of the single-chain antibody with a short peptide chain linker.The mature anti-tumor polypeptide ZXR-2 that has been studied in the laboratory is selected.Designed and synthesized the amino acid sequence of EGFR protein(huscFv-ZXR-2)fused with anti-tumor peptide ZXR-2.And reverse translated to get the nucleotide sequence,humanized optimization of the sequence.And the humanized single-chain antibody huscFv without ZXR-2 is used as a control.The two fragments were digested,ligated,induced expression and purified by genetic engineering method,and the operation parameters in the induced expression and purification process were optimized and screened to obtain the experimental parameters with the highest protein expression and purity.The renaturation conditions of the protein are then optimized to increase the recovery of protein expression.Western blot was used to detect the specificity of the expressed protein.ELISA experiments were performed on the protein to detect the antigen-antibody binding activity.The cell growth inhibition experiment was used to detect the degree of protein growth inhibition of cells with different EGFR expression levels.The results show that the two recombinant vectors huscFv-ZXR-2 and huscFv have been successfully constructed and successfully expressed in E.coli.The induced proteins are all in the form of inclusion bodies.The highest expression level is 16? for 12 h.By comparing the denaturation treatment of inclusion bodies with guanidine hydrochloride and urea and the purification effects of Ni-NTA column and Co-NTA column,the results show that the purification effect of Ni-NTA column is better under the condition of urea denaturation.In the later renaturation process,the urea concentration was 2 M,and the protein purity and yield after gradient dialysis were higher.The results of ELISA experiments showed that both huscFv-ZXR-2 and huscFv proteins can specifically bind to EGFR,and the binding amount of huscFv-ZXR-2 is higher.The results of cytotoxicity experiments showed that the inhibitory effect of the two expressed proteins on different cells was proportional to the expression of EGFR,and huscFv-ZXR-2 showed a stronger inhibitory effect.In short,this study screened out the most suitable expression and purification parameters for the two proteins,and both proteins have specific binding effects on EGFR,and the effect of proteins with anti-tumor peptides is more obvious.