Construction And Identification Of Recombinant HEK 293T Cell Lines Expressing Ectodomain Glycoprotein Of Rabies Virus

BackgroundRabies virus infection remains a serious public health threat in developing countries.The development of a novel and low-cost ELISA diagnostic method is essential for early detection and prevention of the disease.As the main structural protein of rabies virus,rabies virus glycoprotein(G)has important research value in the development of diagnostic kits,virus infection,immune response,and vaccine research.However,the difficulty to express and purify RABV G protein with natural structure is the major bottleneck at present.IntroductionThis study aims at establishing an eukaryotic expression system with stable and robust yield of secretory ectodomain RABV G,which may lay a foundation for the development of ELISA diagnostic kits and novel subunit vaccines.MethodsA recombinant lentiviral expression vector containing ectodomain RABV G gene was constructed and transfected into HEK 293T cells.Culture supernatant was collected 48h hours after transfection and identified by Western Blot.Then,recombinant lentiviral vector LV-CMV-RABV-G-eGFP was packaged and transduced to 293T cells at an MOI of 1000,recombinant cell lines with stable expression of RABV G were selected by limiting dilution and detected by Western Blot and flow cytometry.Recombinant RABV G was purified by affinity chromatography and determined by SDS-PAGE.After that,the concentration of the purified protein was determined by BCA method.Antigenic characteristic of recombinant RABV G was identified by Western Blot and ELISA.For in vivo study,Balb/c mice were immunized by multiple sites subcutaneous injection of purified rRABV G,Several days after immunization,serum samples were collected,the levels of anti-RABV G antibody and neutralizing antibody were detected by ELISA and Fluorescent Antibody Virus Neutralization Test(FAVN),respectively.ResultsThe recombinant lentiviral vector pLV-CMV-RABV-G-eGFP was successfully constructed.Western Blot showed efficient RABV G protein expression in transfected cells and supernatant.Recombinant lentiviral vector LV-CMV-RABV-G-eGFP was transduced to HEK 293T cells and 6 recombinant cell lines were obtained by limiting dilution.Western Blot showed that the No.2 recombinant cell line achieved the highest level of rRABV G expression in the supernatant,which was named HEK-293T-RABV-G-2.Western Blot and flow cytometry results showed that the rRABV G expression level,fluorescence rate,and fluorescence intensity of the recombinant HEK-293T-RABV-G-2 cell line did not significantly change for at least 40 passages.The rRABV G was purified by affinity chromatography,SDS-PAGE showed a purity of up to 95%.Western Blot using anti-RABV G antibody has confirmed the antigenic specificity of the recombinant protein.ELISA results showed that the OD value was strong positive linearly correlated with neutralizing antibody titers.In vivo study showed that high level of anti-G antibody was detected in serum samples of immunized mice.However,the result of FAVN showed that no neutralizing antibody was detected in serum.ConclusionIn this study,a recombinant HEK 293T cell line with stable and high efficiency expression of secretory ectodomain rRABV G was successfully constructed.The recombinant RABV G stimulated the body to produce high levels of anti-G antibodies without any protective neutralizing antibodies.This study laid the foundation for the development of novel serological diagnostic kit and subunit vaccines for RABV.In addition,the established recombinant cell line may provide a cell model for studying the biological characteristics of RABV G.