Preparation Of Monoclonal Antibody Against Zika Virus NS1 Protein And Establishment Of Double-antibody Sandwich ELISA

Zika virus(ZIKV)is an emerging mosquito-transmitted flavivirus,which similar to dengue fever(DENV),Japanese encephalitis(JEV),and West Nile fever(WNV).ZIKV is the single stranded RNA viruses belonging to the flaviviridae family,genus.ZIKV contains a positive-sense 11 kb genome RNA that encodes three structural proteins(C,prM,and E)and seven nonstructural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,and NS5).It was estimated that more than 1.5 million people confirmed infected with the ZIKV and over 46 countries had been reported for the cases of the natural ZIKV infections.Nonstructural protein 1(NS1)is the important protein secreted by the virus and interacts with the host.It forms the homologous dimers in the cell and binds to the type of adipocyte membrane system,participating in viral replication,with the host immune system and other host factors help virus immune escape,and strengthen the interaction between pathogenic.Besides,NS1 as the main antigen can induce the body to produce antibodies,which is an important marker in early diagnosis of ZIKV.Therefore,the development of the detection kits based on the ZIKV-NS1 protein has important practical significance for the clinical diagnosis and treatment of the ZIKV.The main contents and results of this experiment are as follows:Part 1: Expression and purification of the recombinant ZIKV-NS1 protein.A pair of specific primers containing EcoR I and Xho I sites was designed using the primer 5 software,according to the ZIKV-NS1 gene sequence and primer design principle.The target gene was amplified and inserted into the pET-32 a expression vector,and then the positive clones containing the target fragment were confirmed by PCR and DNA sequencing.Next,the pET-32a-ZIKV-NS1 was transfected into E.coli Rosetta(DE3)cells and then the recombinant ZIKV-NS1 proteins were expressed and purified.The results of SDS-PAGE indicated that the recombinant NS1 protein was expressed in large quantity in inclusion body.Thereafter,the precipitations were denatured by 8M urea and purified using nickelnitrilotriacetic acid.Next,the recombinant NS1 proteins were achieved after refolded in PBS buffer.The results of SDS-PAGE and Western blot showed that the recombinant ZIKV-NS1 proteins were expressed and purified successfully and the molecular weight of recombinant protein was 58 KD.Part Ⅱ: Production of monoclonal antibodies(mAbs)against the ZIKV-NS1 protein.The purified recombinant ZIKV-NS1 protein was used to produce antibodies in BALB/c mice by multipoint subcutaneous injections.After the immunizations,the spleen cells of mice were collected and fused with SP2/0 cells.The RPMI 1640 containing 20% FBS and hypoxanthine-aminopterin-thymidine was used to culture fused cells.One week later,the fused cells were cultured in RPMI 1640 containing 20% FBS and hypoxanthine-thymidine.The indirect enzyme-linked immunosorbent(ELISA)and indirect immunofluorescent assay(IFA)were used to determine the presence of the ZIKV-NS1-specific antibodies in the supernatant of hybridoma.Through three cycles of subcloning,fourteen anti-ZIKV-NS1 antibody positive hybridoma cells were prepared successfully.Part Ⅲ: After obtaining the hybridoma cell line,the hybridomas cells were used for the preparation of ascites.The reactivity and specificity of the ascites were evaluated by ELISA,Western blot,and IFA assay.The results of Western blot and IFA revealed that mAbs prepared here could react specifically with the native NS1 protein.Next,all ascites were purified and conjugated with Horseradish Peroxidase(HRP)for the development of the double-antibody sandwich ELISA(DAS-ELISA).The sensitive of this method is 120 ng/mL and can detect the ZIKV in the supernatant and cell lysates of Vero,BHK cells,and the serum of the tree shrew that infected with ZIKV.In this study,a large amount of ZIKV-NS1 protein was expressed and purified.mAbs and pAb specifically binding the native ZIKV-NS1 protein were prepared successfully.Further,the DAS-ELISA was developed based on the group of pAb R1,with the high affinity functioned as the capture antibody,and mAb 1F12 as signal antibody.Without the isolation of the virus and extraction of the RNA,the DAS-ELISA can be used as a primary screening method to improve the detection efficiency of the ZIKV,which provides an effective protection for the control the outbreak and timely treatment.