Generation Of Monoclonal Antibodies Against NS5 Protein Of ZIKV And The Expression Of NS1 In Baculovirus System

Zika virus(ZIKV)belongs to the Flaviviridae family,crossing the blood-brain barrier and enter the central nervous system after infecting the human body,and leading to fetal brain malformation and adolescent Guillain Barre syndrome(GBS).Up till now,there is no effective commercial vaccine or antiviral drug against ZIKV infections.NS5 is the largest non-structural protein of ZIKV,consisting of an RNA-dependent RNA polymerase(RdRp)and a methyltransferase(MTase).It is an essential protein for viral genome replication and also decrease the elicitation of the host's natural immune response.In addition to NS5,NS1 is also a very important multifunctional non-structural protein of ZIKV.It can induce non-neutralizing antibodies but have protective role in infected cells through the antibody-dependent cellular cytotoxicity(ADCC)mechanism and complement dependent pathway,so it is used to develop ZIKV vaccines and therapeutic drugs,and has stronger safety.In this study,monoclonal antibodies were produced against ZKIV NS5 protein along with construction of recombinant baculovirus,expressing NS1 protein.This project was helpful to explore the mechanism of ZIKV replication and vaccine development.The specific research content is as follows: 1.Preparation and identification of ZIKV NS5 monoclonal antibodyRNA was extracted from the ZIKV MR766 strain and then reverse-transcribed into cDNA.Using this cDNA as a template,the NS5 gene sequence was amplified by the polymerase chain reaction(PCR).pET-28 a vector was used to construct prokaryotic expression plasmid(pET-28a-ZIKV-NS5).The expression of ZIKV NS5 protein was induced by using isoform-?-D-1-thiogalactopyranoside(IPTG)and then purified the protein.Four-week-old BALB/c mice were immunized with purified ZIKV NS5 protein.After cell fusion,10 positive hybridoma cell line strains were selected on the basis of indirect enzyme-linked immunosorbent assay(ELISA)and sub-cloning techniques.These strains were named as 1H5,1D3,1D11,2A11,2C1,3A2,3A7,4D11,4F12 and 4G11 respectively that can stably secrete ZIKV NS5 monoclonal antibodies.The results of indirect immunofluorescence Assay(IFA)and Western Blot(WB)indicated that 1H5,2A11,2C1,3A2,4D11,4G11 were only responded to ZIKV but 1D3,1D11,3A7,4F12 not only reacted with ZIKV,but also showed response against DENV2.2.Construction of recombinant baculovirus expressing ZIKV NS1 proteinZIKV NS1 gene was cloned into pFastBac1 to construct baculovirus recombinant plasmid.The recombinant plasmid(pFastBac1-ZIKV-F-NS1)with signal and(pFastBac1-ZIKV-NS1)without signal was transformed into DH10 Bac,and obtained two baculovirus shuttle plasmid.One was rBacmid-ZIKV-F-NS1 and the other one was rBacmid-ZIKV-NS1.After that shuttle plasmid was transfected into Spodoptera Frugiperda(sf9)cells,a recombinant baculovirus that were expressing the ZIKV NS1 protein was generated and amplified.For large scale expression of the recombinant protein,recombinant baculovirus were infected to High5 cells.Supernatant of the culture medium was purified by nickel affinity chromatography to obtain high-purity ZIKV NS1 protein.SDS-PAGE and WB showed that the protein was successfully expressed.This purified ZIKV NS1 protein was used to immunize BALB/c mice.Serum-specific antibody titer assay showed that the purified ZIKV NS1 protein can induce specific cellular and humoral immune responses in mice and has good immunogenicity.