Experimental Study Of RGD-penetrating Peptide-guided Anti-p21Ras Single-chain Antibody In The Treatment Of Colorectal Cancer

Colorectal cancer is one of the malignant tumors with high morbidity and mortality in the world.Supported by the National Natural Science Foundation of China(NSFC)and Yunnan Science and Technology Research Project,our lab constructed intracellular antibodies against p21 Ras in order to block the Ras signal transduction pathway and achieve the goal of targeted therapy for tumors with high expression of Ras gene.However,the expressed single chain antibody protein can not penetrate the cell membrane because of its high molecular weight,so a way to carry single chain antibody to penetrate the cell membrane is urgently needed.RGD short peptide has the function of carrying macromolecule proteins through the tumor cell membrane.Therefore,RGD peptide was chosen as a guide peptide to carry anti-p21 Ras single chain antibody through the tumor cell membrane to detect the mechanism of RGD single chain antibody fusion protein penetrating the membrane and its anti-tumor activity in vitro.Objective: 1.To express RGD-anti-p21 Ras single chain antibody fusion protein and RGD-EGFP green fluorescent fusion protein;2.To detect the penetration mechanism of RGD peptide by labeling RGD peptide with green fluorescent protein;3.To study the effect of RGD-anti-p21 Ras single chain antibody fusion protein on human colorectal cancer cell SW480 in vitro.Finally,RGD peptide carrying anti-p21 ras single chain antibody can penetrate the cell membrane to inhibit human colorectal cancer cell SW480 and explain the mechanism of RGD peptide penetration,providing a new method for targeted treatment of colorectal cancer.Methods: 1.Prokaryotic expression,purification and identification of RGD-antip21 Ras single chain antibody fusion protein: prokaryotic expression of RGD-antip21 Ras single chain antibody fusion protein and purification,Western Blot and ELISA assay detection of RGD-anti-p21 Ras single chain antibody fusion protein and antigen Ras immune response,and the introduction of protein linker between RGD peptide and single chain antibody to detect linker's addition to the antigen Ras.Influences of immune response.2.Membrane penetration test:(1)The ability of RGD-anti-p21 Ras single chain antibody fusion protein to penetrate the membrane was detected by immunohistochemistry and immunofluorescence assay.(2)RGD peptide was labeled with green fluorescent protein EGFP to detect the time and concentration dependence of RGD peptide,the targeting ability of RGD peptide,the effect of low temperature on RGD peptide penetration,the effect of endocytosis inhibitors on RGD peptide penetration,and the effect of changes in extramembrane potential on RGD peptide penetration.3.In vitro experiments: To detect the effect of RGD-anti-p21 Ras singlechain antibody fusion protein on SW480 tumor cells: MTT assay,plate cloning,cell scratch assay and TUNEL apoptosis detection were used to detect the changes of killing,proliferation,migration and apoptosis of tumor cells after RGD-anti-p21 Ras singlechain antibody fusion protein.Results:The fusion protein of RGD-anti-p21 Ras single chain antibody could react with antigen Ras by ELISA and Western Blot assay.Moreover,the linker connection between RGD peptide and single chain antibody did not affect the immune response of the fusion protein.The ability of RGD-anti-p21 Ras single chain antibody fusion to penetrate the membrane was tested by immunohistochemistry and immunofluorescence experiments.The targeting ability of RGD peptide was confirmed by EGFP green fluorescent protein labeling.The RGD peptide fusion protein was time-concentrationdependent.Low temperature could affect the penetrating effect of RGD peptide fusion protein.It was because the endocytosis of RGD peptide was affected by 4 C temperature,which proved that RGD peptide was a kind of tumor targeting protein.Intraspecific endocytosis mechanism.In addition,the incorporation of various cytotoxic inhibitors also affected the penetration effect of RGD peptide fusion protein.It can be concluded that RGD peptide fusion protein is an endocytotic penetration mechanism.In vitro,scratch test showed that RGD-anti-p21 Ras single chain antibody fusion protein could effectively inhibit the migration of SW480 cells.Plate cloning experiments showed that RGD-anti-p21 Ras single chain antibody fusion protein could inhibit the proliferation of SW480 cells.MTT assay showed that the fusion protein of RGD-anti-p21 Ras single chain antibody had certain killing effect on SW480 cells.TUNEL apoptotic assay showed that RGD-anti-p21 Ras single chain antibody fusion protein could significantly promote the apoptosis of colorectal cancer SW480 cells.Conclusions: 1.The fusion protein was purified by nickel ion purification column,and the concentration of the fusion protein was detected by BCA method.The concentration of the fusion protein optimized by codon was higher than that not optimized.2.The immune reaction between RGD-anti-p21 Ras single chain antibody and Ras was verified by ELISA and immunohistochemical experiments.The linker connection between RGD peptide and single chain antibody protein did not affect the antigen Ras and Ras.The immune response of RGD-anti-p21 Ras single chain antibody fusion protein;3.The targeting of RGD peptide and the time-dependent concentration of RGD peptide were confirmed by labeling RGD peptide with green fluorescent protein tracer,while the incorporation of hypothermia and cytotoxicity inhibitors confirmed that RGD peptide is an energy-independent transmembrane mechanism of endocytosis;4.It was confirmed by MTT,plate cloning,cell scratch and TUNEL apoptosis in vitro.It was confirmed that RGD-anti-p21 Ras single chain antibody fusion protein could effectively inhibit the growth,proliferation,migration and apoptosis of SW480 tumor cells.This study provides a new method for the treatment of colorectal cancer by carrying single chain antibody with RGD peptide.