Construction Of Recombinant Influenza Virus Expressing CTLA-4 Antibody And Its Oncolytic Effect

Part ? Construction and screening of recombinant influenza virus expressing CTLA-4 antibodyBackground:Hepatocelluar carcinoma(HCC)is one of the world's malignant tumors,with the world'shighest mortality rate and the second highest mortality rate in Asia.Although.scientists continue to explore ways to treat tumors,the cure rate has not increased significantly due to the rapid growth of tumor cells and the tendency to invade surrounding tissues.Therefore,finding new ways to treat liver cancer has always been a challenge for humans.More and more studies have shown that oncolytic virus can selectively target cancer cells without causing serious adverse reactions in cancer patients.Cytotoxic Tlym-phocyte associated antigen-4(CTLA-4)cantarget the regulatory pathway in T cells and enhance the antitumor immune response,the oncolytic virus expressing CTLA-4 antibody has been studied.Become a hot spot in cancer treatment research.Objective: To rescue and express a recombinant influenza virus expressing the immune checkpoint mu CTLA-4 antibody.Methods:(1)Design of heavy chain/light chain fragments of p HW191-PB1,p HW194-PA and antibody,plasmid recombination and construction of antibody heavy chain of mu CTLA-4 on PB1 of A/Puerto Rico/8/34(PR8)influenza virus The antibody light chain of mu CTLA-4 was constructed on PA,and co-transfected with COS-1 and MDCK cells with 6 plasmids p HW191-PB2,p HW194-HA,p HW195-NP,p HW196-NA,p HW197-M,p HW198-NS of influenza virus PR8.The virus solution was collected,and the virus solution was inoculated into SPF chicken embryo,ultrafiltration concentration,purification and the like to obtain a recombinant oncolytic influenza virus,which was naas r Flu-mu CTLA-4 antibody.(2)Observing r Flu-mu CTLA-4 by electron microscopic observation of the morphology and size distribution of recombinant oncolytic influenza virus r Flu-mu CTLA-4,hemagglutination test,EID50,PCR,etc.(3)Purification of recombinant oncolytic influenza virus r Flu-mu CTLA-4 After concentration,the content of antibody in anti-CTLA-4 was determined by ELISA.Results:(1)Transmission electron microscopy showed that r Flu-mu CTLA-4 was mostly spherical and had spinous processes on the surface.The particle diameter was between 80 and 120 nm,indicating the virus morphology and particle size distribution of recombinant oncolytic influenza virus r Flu-mu CTLA-4.Conforms to the typical characteristics of influenza virus;(2)Collect the positive allantoic fluid,the hemagglutination titer of r Flu-mu CTLA-4 is 29-10,and the EID50 of r Flu-mu CTLA-4 is 10-8 EID50/1m L.The PB1 and PA gene fragments of recombinant oncolytic virus r Flu-mu CTLA-4 were consistent with the expected size.(3)Chicken embryos inoculated with r Flu-mu CTLA-4 virus for 2?3 and 4 days,respectively,and purified by protein,can detect anti-CTLA-4 antibody.Conclusion: In this study,we successfully constructed an influenza virus expressing the immune checkpoint inhibitor CTLA-4 antibody,and initially completed the identification of the virus,providing a basis for the next in vitro and in vivo experiments.Part ? In vitro and in vivo studies of recombinant influenza virus expressing CTLA-4antibody in the treatment of H22 hepatoma cellsObjective: The therapeutic effect of r Flu-mu CTLA-4 constructed on mouse H22 liver cancer was evaluated.Methods:(1)In vitro experiments,using MTS method to detect the killing effect of oninfluenza virus r Flu-mu CTLA-4 on H22 cell line,and detecting the cytotoxicity of r Flu-mu CTLA-4 on H22;(2)in vivo experiment: establishing Balb/c mouse liver cancer.The H22 cell implantation model was used to observe and evaluate the inhibitionand prolongation of tumorigenesis by the recombinant oncolytic influenza virus r Flu-mu CTLA-4.(3)After treatment of the mice,blood was taken from the eyeballs,serum was collected,and protective antibodies produced by the mice were detected by hemagglutination inhibition test.Results: MTS assay showed that 1.0 MOI recombinant virus P=0.0307(P<0.05),2.0MOI recombinant virus P=0.0017(P<0.01),5.0 MOI recombinant virus P=0.0011(P<0.01),2.0 MOI,The activity of 5.0 MOI virus was significantly lower than that of 1.0 MOI group in a dose-dependent manner,indicating that r Flu-mu CTLA-4 can specifically kill H22 cells(P<0.05).In the mouse model,tumor volume can be significaninhibited and the survival of the mouse is prolonged.The hemagglutination inhibition(HI)assay detected a higher hemagglutination inhibition antibody titer.The experi-mental results indicated that the recombinant virus r Flu-mu CTLA-4 can induce protective antibodies in mice.Conclusion: r Flu-mu CTLA-4 can kill and kill liver cancer cells in vitro and in vivo,and can inhibit the growth of tumor volume in mice,prolong the survival of mice,and protect the body of mice after virus treatment.This study is expected to dissolve.Tumor virus targeted therapy for liver cancer provides new ideas.