Generation Of Monoclonal Antibodies Against Gn1 Protein Of RVFV And The Expression In Baculovirus System

Rift Valley Fever Virus?RVFV?belongs to the genus Phlebovirus of the Bunyaviridae family.It is transmitted by mosquitoes and can cause Rift Valley Fever?RVF?,a febrile zoonotic disease,resulting in a large number of deaths of ruminants and fatal hemorrhagic fever in humans.RVF was only prevalent in East Africa at first,but with the increase of import and export trade,it has gradually expanded in the global scope.In 2016,the first imported case in China of RVF was confirmed.Currently,the vaccine used to prevent the RVFV epidemic is only partially attenuated,which is expensive and induces short-term immunity,and there are no specific treatment options for infection.RVFV capsule membrane protein?G?was lyzed into Gn protein and Gc protein during the post-translational modification process.Gn protein can induce the host to produce neutralizing antibody,so it is an important target for the study of genetic engineering vaccine.In this study,the selected antigen region Gn1 was determined from the main antigen region of Gn protein.The monoclonal antibody against RVFV Gn1protein was prepared and the baculovirus expression system was used to construct the recombinant baculovirus expressing Gn1 protein,so as to provide important materials for RVFV vaccine research and development.The specific research content is as follows:1.Preparation of monoclonal antibody against RVFV Gn1 proteinThe selected Gn1 fragment was synthesized as a cDNA template and its gene sequence was amplified by PCR.Then the target fragment was cloned into the prokaryotic expression vector pET-28a to construct the prokaryotic expression plasmid pET-28a-RVFV Gn1.At 37?,with the concentration of 1.0 mM/L IPTG induction and inclusion purification,RVFV Gn1 protein was obtained.Purified RVFV Gn1 protein immunized 4-week BALB/c mice.Through cell fusion,ELISA assays and three times of subcloning,2 strains of positive hybridoma cell lines were selected and named as:6D3and 11B8,respectively,which were capable of secreting stable RVFV Gn1 monoclonal antibody.Indirect immunofluorescence and Western Blot validation were used to verify that both of them could react with RVFV Gn1 eukaryotic and prokaryotic proteins,and the antibody titer was 1:819200,1:1638400.The analysis of subtypes of the three monoclonal antibodies showed that the heavy chains were IgG_2b,and the light chains were all Kappa.2.Construct the recombinant baculovirus expressing RVFV Gn1 proteinRVFV Gn1 gene was cloned into baculovirus vector pFastBac1/HTB to construct baculovirus recombinant plasmid.The recombinant plasmid pFastBac1/HTB-RVFV-F-Gn1 with signal peptide and the recombinant plasmid pFastBac1/HTB-RVFV-Gn1without signal peptide were transformed into DH10Bac receptor,and the shuttle plasmid rBacmid-RVFV-F-Gn1/rBacmid-RVFV-Gn1 was obtained.The shuttle plasmid was transfected into sf9?Spodoptera Frugiperda,sf9?cells,and the recombinant baculovirus expressed in the supernatant,proved by IFA assays.Infecting High5 cells with the baculovirus,the recombinant protein expressed and was purified by nickel affinity chromatography.Through SDS-PAGE and Western Blot analysis,we found the RVFV Gn1 protein with high purity highly expressed in cell lysis supernatant.In this study,we obtain RVFV Gn1 recombinant prokaryotic protein,the main antigen region of Gn protein of rift valley fever virus and 2 strains of monoclonal antibodies,named 6D3 and 11B8 through immunization,fusion and subclonal.We also successfully construct the recombinant baculovirus of RVFV Gn1 and the recombinant eukaryotic protein highly expressed in the supernatant of cell lysis.