Lentiviral Vectors-mediated High Efficiency Expression Of Recombinant Humanepidermal Growth Factor In HEK-293T Cells And Activity Determination

BackgroundEpidermal growth factor(EGF)is a 6-kDa protein with 53 amino acid residues and three intramolecular disulfide bonds.EGF acts by binding with high_affinity to epidermal growth factor receptor(EGFR)on the cell surface,which results in cellular proliferation,differentiation,and survival.EGF was originally described as a secreted peptide found in the submaxillary glands of mice and in human urine.EGF has since been found in many human tissues including submandibular gland,parotid gland and in urine,saliva,milk,and plasma.EGF is widely used in medical treatment of skin trauma,postoperative wounds,acne,mouth ulcers and gangrene,and dermatitis caused by radiation therapy.It can accelerate the healing of wounds and ulcers,promote skin metabolism and reduce skin deformities,and can be used as a cosmetic additive and used for facial plastic surgery.EGF is a very important growth factor for human endocrine.However,EGF currently used in clinic is mostly obtained through genetic engineering synthesis,mouse submandibular gland extraction and prokaryotic expression,etc.,which is expensive and biologically unstable.Therefore,it is very important to establish a new type of EGF production system with stable and high efficiency protein yield.ObjectivesThis project aim to establish a recombinant hEGF expressing system in mammalian cells.Secretory-fusion vector with hEGF and IgG Fc fragment was constructed,which can simplified the purification process and increase the stability and plasma half-life of recombinant hEGF.MethodsThe hEGF-IgG gene was cloned into the lentiviral vector pLV-CMV-eGFP.Recombinant plasmid pLV-CMV-Fc-hEGF-eGFP was verified by double digestion and DNA sequencing.The lentiviral vectors were packaged adopting the three-plasmid packaging system and transfected to HEK-293T cells at a MOI of 1000.The transduced cells were then clonally expanded by limiting dilution,two to three weeks later,clones in good condition were picked and cultured.Gene expression of the recombinant cell lines was evaluated by Western blot using an anti-hEGF polyclonal antibody.Recombinant hEGF in culture supernatant was collected and purified by affinity chromatography.Finally,the purity of rhEGF was investigated by SDS-PAGE and the biological activity was evaluated in NIH3T3 cells and Balb/c trauma models.ResultsResults showed that the recombinant plasmid pLV-CMV-Fc-hEGF-eGFP was successfully constructed.HEK-293T cells were infected with lentiviral vector LV-CMV-Fc-hEGF-eGFP,and 8 recombinant HEK-293T cell lines with stable expression of recombinant hEGF were obtained.Western Blot analysis showed that the 4th clonal cell line achieved the highest expression level of Fc-hEGF and lasted for more than 9 weeks in a stable level,which was named HEK-293T-Fc-hEGF SDS-PAGE showed the protein purity of Fc-hEGF was up to 90%.Biological activity study confirmed that Fc-hEGF could promote cell proliferation in NIH3T3 cells and wound healing in Balb/c trauma models.ConclusionIn this study,we have successfully established a robust and stable expression system for recombinant hEGF in eukaryotic cells,which may lay a foundation for the application of EGF.