Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody

Abstract

Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

“…In vitro transcription and cell-free protein synthesis were performed using WEPRO7240 wheat germ extract (CellFree Sciences, Yokohama, Japan). Transcription was performed using SP6 RNA polymerase, as described previously ( Matsunaga et al, 2014 ). The translation reaction was performed in bilayer mode with slight modifications.…”

Engineering Cellular Biosensors with Customizable Antiviral Responses Targeting Hepatitis B Virus

“…Immunization of BALB/c mice with full-length recombinant LHBs protein (Beacle. Inc.) and generation of hybridoma cells producing α-HBs antibody were performed as previously described (Matsunaga et al, 2014;Yamaoka et al, 2016).…”

Hepatitis C virus infection suppresses hepatitis B virus replication via the RIG-I-like helicase pathway

“…Cells were washed again (3× PBS) and blocked in PBS/0.2% BSA. HBc antigen monoclonal antibodies were generated by immunizing of BALB/c mice with recombinant HBc protein synthesized in a wheat cell-free protein production system, as previously described 29 . HBc antigen was probed by using a mouse monoclonal antibody against HBc antigen (clone 7B2, culture supernatant of the hybridoma) 30 diluted 100-fold in PBS (1 hour at room temperature).…”