The Establishment Of An Indirect ELISA Detection Method And Preparation Of Monoclonal Antibodies Against Equine Hendra Virus Nucleocapsid Protein And Glycoprotein

Hendra virus(HeV),a member of the genus Hennipavirus family Paramyxoviridae,is a rare but dangerous zoonotic pathogen,which characterized by severe respiratory symptoms and high mortality in horses.Until 2021,65 cases of HeV infection in horses have been officially reported in Australian,resulting in the death of 105 horses and posing a huge risk to the equine breeding industry.Symptoms of HeV infection are similar to those other respiratory diseases,so it is important to establish accurate laboratory diagnostic methods for HeV.Although RT-PCR is the diagnostic technique that laboratory advice for clinical case confirmation,which can detect HeV in a biosafety level-2 laboratory,it is challenging to evaluate the history of HeV exposure in the animals to be examined.Thus,serological detection procedures,in addition to RT-PCR,are required.HeV is a negative single-stranded RNA virus,encoding six structural proteins.G protein is the sole attachment glycoprotein exposed on the external surface of envelope viruses,and infects cells through Ephrin B2/3 receptor binding to host cell,and initiates the host cell phagocytosis process together with F protein,resulting in delivery of the nucleocapsid into the cytoplasm,which is a vital anchoring protein and target of neutralizing antibody in the infection pathway.The N protein,which wraps the viral genome,is the most abundantly expressed nucleocapsid protein,and its C-terminal domain interacts with the P protein to form the N-P complex,which binds RNA polymerase and supports viral replication and transcription.N protein is released into the cytoplasm after fusion of the viral envelope and host cell membrane,and the N protein is immunogenic.Both are effective targets for the development of serological assays for HeV.Based on the prokaryotic recombinant proteins of HeV-N and HeV-G,an indirect ELISA(i ELISA)approach for detecting antibodies against aquine-derived HeV was establishment in this work,and mouse monoclonal antibodies(m Abs)was produced.The specifics are as follows:1.Synthesis and expression of prokaryotic recombinant HeV-N and HeV-G proteins:In this investigation,the HeV(NC＿001906.3)N and G genes were codon optimized for efficient expressed in the Escherichia coli(E.coli)system.The synthetic target genes were cloned into the p ET-30a(+)prokaryotic expression vector to obtain HeV-p ET30a(+)-N and HeV-p ET30a(+)-G recombinant plasmids.They were transformed into BL21(DE3)strain for chemically induced expression following proper sequencing.Purified recombinant proteins were obtained through the identification of SDS-PAGE and Western blot,as well as purification by nickel affinity chromatography.2.Establishment of i ELISA test method:Horses were immunized with 100μg of recombinant protein per dose.After 3immunizations,the horse serum was harvested.The recombinant antigen was coated on ELISA plates.Horse serum after immunization was used as the positive control,whereas the negative control was other serums with RT-PCR negative and no vaccination history.Ultimately,it was determined that HeV-N and HeV-G protein coating doses were determined to be 1μg and 0.8μg,respectively,the serum dilution was 1:1 000,and the critical values were 0.235 and 0.267.The sensitivity of limiting dilution method to determine HeV-N and HeV-G positive serum were higher than 1:12 800 and 1:6 400,respectively.The assay showed no cross-reaction with 9known antibodies positive serum against other equine pathogens,and has good specificity.The coefficient of variation was less than 10%,which were proved by intra-batch and inter-batch repeated tests.As the data published by China Animal Health and Epidemiology Center,the results of 631 clinical equine samples evaluated using this assay from various countries were all negative.The above results indicated that the i ELISA method established in this experiment can be invoked as a serological technical reserve for clinical detection of HeV antibodies.3.Preparation of mouse polygonal antibody and m Abs:BALB/c mice were immunized with recombinant protein at dose of 100μg/per.After 4immunizations,high-titer serum antibodies against antigens specific reaction were harvested as the positive control.In this experiment,the HeV N and G genes were cloned into pc DNA3.1(+)eukaryotic expression vector.The pc DNA3.1-N-His and pc DNA3.1-G-His plasmids were successfully constructed.Mice with high-titer antibodies were selected to dissect splenic lymphocytes,eventually,three m Abs against HeV-N(2H6,3C2,3G6)and two m Abs against HeV-G(5B8,2A4)proteins were obtained and screened out via classical hybridoma technology and identified by i ELISA,Western-blot and IFA.The biological characteristics test results suggested that the titer of antibodies were more than 10~5all,and they were likewise fine responsed to recombinant proteins.The result of subtype identification showed that 2H6,3C2and 3G6 were Ig G2b,and the heavy chains of 5B8 and 2A4 were Ig G1.In summary,this experiment successfully established an i ELISA assay based on HeV-N and HeV-G proteins,which provides a new idea for the clinical detection of HeV infection,and produced m Abs against the proteins to offer direction for future research on HeV detection targets,along with the development of vaccinations.