Generation Of Monoclonal Antibodies Against GP Protein Of EBOV And Its Research In Baculovirus Expression System

Ebola virus disease(EVD),as known as Ebola hemorrhagic fever(EHF), is a severe viral hemorrhagic fever caused by Ebola virus(EBOV) which belongs to Filoviridae family. The 2014-2015 Ebola epidemic in Western Africa has been the most protracted and devastating in the history of this disease. GP belongs to type I transmembrane protein and play multiple roles in virulence, viral infection and induction of neutralizing antibody activity. In this study, specific monoclonal antibodies against EBOV GP were produced and the EBOV GP protein is also expressed in the Bac-to-Bac Baculovirus Expression System. Also, the immunogenicity of GP protein was investigated through animal experiment. This study set foundation for EBOV vaccine and nosogenesis research. Main content of this study is as follows:1. Preparation and identification of monoclonal antibodies against EBOV GPProkaryotic expression vector pGEX-KG with the front 300 aa of EBOV GP protein was constructed with the analysis of DNA Star. After transformation, induction, expression and inclusion body purification, the purified truncated GP protein was obtained. BALB/c mice were immunized with the protein. Through cell fusion and ELISA, four strains which can steadily secrete antibody against EBOV GP were acquired, called 1C1、2A3、2G7、2H9. The IgG subtype and light chain of these monoclonal antibodies(McAbs) were IgG1 and Kappa. By IFA and Western blot using recombinant baculovirus, all of four subclones strains were specific and sensitive.2. Construction recombinant baculovirus with GP gene and the research of GP’S immunogenicityThe EBOV GP gene was amplified directly from plasmids contain this gene, then this gene fragment was inserted into donor plasmid pFast Bac HT B. By specific gene transposition, the recombinant Bacmid-EBOV-GP contains GP gene was acquired. By transfected spodoptera frugiperda cell, the recombinant baculoviruses BV-EBOV-GP was rescued successfully. The results of molecular biology experiments(IFA and Western blot) showed the EBOV GP gene could be expressed correctly. Then BALB/c mice were immunized with that protein and specific antibody titer in serum and lymphocyte proliferation was observed. The experiment results show that recombinant Bacmid-EBOV-GP has good immunogenicity.