Preparation And Activity Analysis Of AD? Fused With Cell-penetrating Peptide

So far,the overall mortality rate associated with cancer is still at a relatively high level.Strict cell cycle regulation is the functional guarantee for the body to carry out normal physiological activities,and it generally shows that cell cycle regulation is out of control in human cancer.Cyclin D1 is an important driver of the cell cycle.Overexpression and activation of cyclin D1 occur in a variety of tumor cells.In recent years,more and more evidence has shown that using cyclin D1 as a target for tumor diagnosis and treatment is highly feasible.Single-chain antibodies show great potential in tumor therapy due to their high specificity,small molecular weight,and ease of engineering modification.In the previous work,our research group successfully screened and prepared a single-chain antibody AD? that can specifically recognize and inactivate cyclin D1 in cells.and has good antitumor effects in vivo and in vitro.However,the single-chain antibody AD? cannot effectively penetrate the cell membrane,making it difficult to act on target molecules in the cell,thereby greatly limiting its application.In recent years,the more efficient cell-penetrating peptides that can be used as delivery vehicles have been studied.Their efficient and low-toxic membrane-penetrating ability has provided a new way for the transmembrane delivery of therapeutic drugs,as well as new ideas for antibody protein transmembrane therapy.To this end,on the basis of the single-chain antibody AD? prepared in the previous stage of the research group,the cell penetrating peptide(PTN)and TAT were connected to its C-terminus to construct a novel recombinant human AD?protein AD? with a penetrating peptide sequence AD?-PTN and AD?-TAT two prokaryotic expression vectors,induced expression and purification of them to obtain recombinant fusion proteins AD?-PTN and AD?-TAT containing cell transmembrane peptides,ELISA analysis found that the new recombinant fusion protein is relatively different from Cyclin D1 Good affinity activity,so as to lay the foundation for anti-tumor treatment of single-chain antibody AD?,and promote the further development of antibody tumor clinical treatment.The results are as follows:1.Using the plasmid of the anti-Cyclin D1 single chain antibody(AD?)prepared successfully in the early stage of the laboratory as a template,the cell penetrating peptides PTN and TAT were cloned into the C terminus of the single chain antibody AD? by PCR reaction to obtain New recombinant human AD? gene fragments AD?-PTN and AD?-TAT,construct recombinant prokaryotic expression vectors and prepare secreted expression bacteria pDF-AD?-PTN/HB2151 and pDF-AD?-TAT/HB2151,after induction by IPTG The supernatant of the culture medium was analyzed by ELISA,and it was determined that the modification of penetrating peptide and TAT did not affect the antigen binding activity of AD?.2.In order to obtain a large number of recombinant proteins fused with cell-penetrating peptides PTN and TAT,AD?-PTN and AD?-TAT were cloned into pET28 b vectors respectively,and recombinant expression vectors pET28b-AD?-PTN and pET28b-AD?-TAT were successfully constructed.Expression bacteria pET28b-AD?-PTN/BL21(DE3)and pET28b-AD?-TAT/BL21(DE3)for recombinant AD? fused with cell-penetrating peptides were prepared successfully.3.At 0.5 mM IPTG and 30?,the target protein AD?-PTN expression was highest for 4 h,while for AD?-TAT at 1 mM IPTG and 30?,the highest expression was induced for 4 h,The recombinant fusion proteins AD?-PTN and AD?-TAT had Soluble and inclusion bodies are two expression forms and most of them exist as inclusion bodies.4.Using 8M urea denaturation and His Trap HP affinity chromatography to purify the AD?-PTN and AD?-TAT inclusion bodies,the optimal imidazole elution concentration of the target protein is 100 mM,and the eluent is successfully renaturated by dilution and then desalted to obtain AD?-PTN and AD?-TAT proteins.5.The purified recombinant fusion proteins AD?-PTN and AD?-TAT were identified by ELISA and Western blot,indicating that the transmembrane peptide fusion AD?-ADN and AD?-TAT with high biological activity were successfully obtained.6.The ELISA results of the antigen-binding activity of the purified AD?-PTN and AD?-TAT proteins showed that the purified recombinant proteins AD?-PTN and AD?-TAT could bind cyclinD1 with high specificity,and there was no significantdifference in the binding activity of AD?-PTN or AD?-TAT to cyclin D1 compared with AD?.The recombinant proteins AD?-PTN and AD?-TAT fused the cell-penetrating peptides PTN and TAT to the anti-cyclin D1 single chain antibody AD? are laid foundation for the anti-tumor therapy of AD? antibody protein based on the intracellular induction or tissue-specific cyclin D1 knockout.In addition,it also provides a theoretical basis and new ideas for antibody treatment of cancer targeting cyclin D1.