Cell-penetrating Peptide Conjugates Of NFKBIA Mutation Enhance The Antitumor Effect On CRC

Objective:Colorectal malignant tumor is one of the most common malignant tumors in the digestive system.In recent years,the morbidity and mortality have increased significantly around the world.Although modern diagnosis and treatment technology has developed rapidly,the overall curative effect is still limited.The activation of RAS signaling pathway is closely related to the malignant tumors.The subsequent treatment effect and prognosis of patients with K-ras mutant colorectal cancer were significantly lower than others.Studies have shown that the abnormal accumulation of IkBa in the nucleus can inhibit K-ras mutant tumor cells.This project uses gene splicing technology,DNA recombination technology and recombinant protein expression technology,focusing on sequence design,construction of transfection plasmids,preparation and purification of recombinant protein CPP2-DN-NFKBIA-NLS,and research on the target recombinant protein in colorectal cancer cells The metastasis in the system has anti-tumor effects on cell proliferation,migration,and apoptosis.Method:1.Query the target gene sequence and related information through the relevant website,design and optimize the recombinant protein CPP2-DN-NFKBIA-NLS gene sequence,after subcloning,select the prokaryotic expression vector pET28+plasmid in Escherichia coli C41 active bacteria and transfer it into E.coli BL21(DE3),to obtain the recombinant expression.IPTG was used to induce E.coli to express recombinant protein,and to find the best expression conditons of the recombinant protein.Use his-tag affinity purification and SDS-PAGE identification.The recombinant protein DN-NFKBIA-NLS of the control group was designed and obtained by the same method and verified.2.Immunofluorescence technology was used to study the transfer of recombinant protein CPP2-DN-NFKBIA-NLS in colorectal cancer cell line.3.The effects of recombinant protein CPP2-DN-NFKBIA-NLS on colorectal cancer cell lines were detected by MTT method,flow cytometry and scratch method,and the anti-tumor effect of recombinant protein was exploredResult:1.The target protein CPP2-DN-NFKBIA-NLS and the recombinant protein DN-NFKBIA-NLS were successfully constructed and expressed,and the expression conditions of the target protein were clarified.2.The immunofluorescence experiment targeting recombinant protein CPP2-DN-NFKBIA-NLS can enter the nucleus of colorectal cancer cells.3.The MTT experiment proved that the recombinant protein CPP2-DN-NFKBIA-NLS can inhibit the proliferation of various colorectal cancer cell lines;the flow cytometry apoptosis experiment proved that the recombinant protein CPP2-DN-NFKBIA-NLS can promote the proliferation of colorectal cancer cells.Apoptosis;scratch experiments showed no sgnificant effect of the recombinant protein CPP2-DN-NFKBIA-NLS on the migration ability of colorectal cancer cell lines.Conclusion:The recombinant protein CPP2-DN-NFKBIA-NLS was successfully constructed using gene recombination technology and recombinant protein expression technology,and in subsequent experiments it was demonstrated that the recombinant protein has good antitumor activity in colorectal cancer cell lines.Studying the pathogenesis and targeted therapy of colorectal cancer provides new ideas.