Construction And Functional Identification Of Recombinant Adenovirus Encoding EGFRvⅢ Intracellular And Extracellular Regions

Epidermal growth factor receptor（EGFR）expression is elevated in different forms of cancer and positively correlates with cancer progression and poor prognosis.EGFRv III is an active mutant form of EGFR that drives the growth of a variety of malignancies,making it a promising receptor for small molecule targeted therapies.In this experiment,total RNA was first extracted from human prostate cancer cell line PC-3 cells,reverse transcribed to c DNA,and EGFRv III intracellular and extracellular region gene was amplified using c DNA as template,and p Ad Track-CMV plasmid vector was ligated to transform E.coli BJ5183 receptor cells containing p Ad Easy-1 to obtain homologous recombinant plasmid transfected with HEK293A cells.The recombinant adenovirus expressing EGFRv III intracellular and extracellular region protein was obtained.The recombinant adenovirus was then used to immunize mice in high and low dose groups.Two weeks after the second immunization,blood was collected from the eyes and the Ig G antibody level in the serum was measured by ELISA.The results showed that:（1）.The EGFRvⅢ recombinant plasmid was successfully constructed and transfected with HEK293A cells,and the EGFRv III gene was normally expressed in HEK293A cells by PCR and Western-blot.（2）.The titer of recombinant adenovirus was determined by TCID50 method after purification and concentration,and the titer of recombinant adenovirus was calculated as 1.7×10¹¹PFU/m L for intracellular region,1×10¹⁰PFU/m L for extracellular region,and 1×10¹¹PFU/m L for CMV recombinant adenovirus.（3）.After immunization of mice with recombinant adenovirus,the levels of Ig G antibodies in mouse serum were 1:800 in the intracellular group and 1:1600 in the extracellular group.The levels of IFN-a and IL-2 in mouse serum samples were determined by ELISA kits.The results showed that the levels of IFN-a and Th1-type cytokine IL-2 in the serum of mice after immunization were significantly higher than those in the control group,P<0.01,which was statistically significant.The results showed that the recombinant adenovirus expressing EGFRv III intracellular and extracellular region gene was successfully constructed,and the recombinant adenovirus could induce specific humoral immune response in mice.