[Preparation of monoclonal antibody against enterovirus 71 capsid protein VP1].

Objective To prepare murine monoclonal antibody (mAb) against enterovirus 71 (EV71) capsid protein VP1. Methods VP1 protein was expressed and purified. BALB/c mice were immunized with inactivated and purified EV71 virus, and mAb specific to EV71 VP1 was generated by hybridoma technique. Indirect ELISA was used to test antibody titer and antibody subclass identification. The expression of VP1 protein was detected by Western blot in EV71-infected RD cells. The expression and distribution of VP1 protein was detected by immunofluorescence cytochemistry in EV71-infected RD cells. Results Six antibody strains were obtained, among which three were IgG2a and three were IgG2b, all of which could be used for ELISA, Western blot and immunofluorescence cytochemical staining. 2D7 exhibited neutralization capacity with 50% inhibitory concentrations(IC50) of 9.892 µg/mL. Conclusion Six strains of monoclonal antibodies with excellent reactivity were obtained, which laid a foundation for the further studies on the identification and diagnosis of EV71 as well as the functional of VP1 protein.

[Establishment and identification of human hepatocellular carcinoma line stably expressing hepatitis C virus core protein].

OBJECTIVE: To establish SMMC-7721 human hepatocellular carcinoma cell line stably expressing hepatitis C virus (HCV) core protein. METHODS: A lentiviral vector containing HCV core gene was constructed and transfected into HEK293T cells to package recombinant lentivirus (rLV-core) containing ZsGreen and HCV core genes. The SMMC-7721 cells were infected with the rLV-core. The expression of HCV core mRNA was examined by real-time fluorescent quantitative PCR and the HCV core protein was detected by immunofluorescence cytochemistry and Western blotting. The stably transfected cell line was screened. RESULTS: The lentiviral vector was confirmed by enzyme digestion and sequencing. The green fluorescence was seen under fluorescence microscope 48 hours after virus packaging. The SMMC-7721 cell line stably expressing HCV core protein was obtained after infected with the rLV-core. Real-time PCR showed the expression of HCV core mRNA, and both immunofluorescence cytochemistry and Western blotting verified the expression of HCV core protein. CONCLUSION: The SMMC-7721 human hepatocellular carcinoma cell line stably expressing HCV core protein has been established successfully.