| Nipah virus(NiV)broke out in Malaysia in 1998 originally,causing fatal encephalitis and severe respiratory diseases in humans.NiV has become a public health concern due to its characteristics of strong pathogenicity,wide host range and potential threat of pandemic.As of June 2018,NiV has caused 643 laboratory confirmed cases,and resulted in at least 380 deaths.At present,there are no effective anti-NiV drugs and vaccines,and in order to prevent the occurrence and epidemic of NiV in China,it is particularly necessary to establish NiV detection methods.Methods:In this study,primers and probe were designed based on the conservative region of NiV N gene to establish the qRT-PCR detection method,and the detection method was evaluated and applied.For the F and G proteins that play a key role in NiV invasion into host cells,NiV pseudovirus was constructed by lentiviral expression vector.NiV pseudovirus was identified and applied to serum neutralizing antibody detection and titer evaluation.NiV N protein was expressed by insect baculovirus expression system,and the recombinant protein was identified,which lays a foundation for further screening of monoclonal antibodies and establishment of NiV antigen detection method.Results:1.The establishment and application of Taqman qRT-PCR detection method for NiV(1)The NiV Taqman qRT-PCR detection method was established.The primers and probe were sepecific,the detection limit was 102 copies/μL,and the linearity of the standard curve allowed quantification of 1.0×109 to 1.0×102 RNA transcripts,R2=0.995 showed a good linear relationship,the coefficient of variation was less than 5%which indicated good stability and repeatability.(2)NiV Malaysian strain and Bangladesh strain can be detected effectively by the detection method.83 samples of lung tissue and brain tissue homogenate of bats were detected by this method,and the results were all negative2.Preparation of NiV pseudovirus and establishment and evaluation of serum neutralization antibody detection method(1)NiV F and G genes were optimized and synthesized,and NiV membrane proteins F and G were expressed by eukaryotic expression vector pCDH-CMV-MCS-EFl-copGFP-T2A-Puro.NiV pseudoviruses were successfully packaged in 293T cells with the help of human immunodeficiency virus vector pNL4-3.Luc.R-E-,and the packaging conditions were optimized.Western Blot confirmed the presence of F and G proteins in the cell lysate.NiV pseudoviral particles were round or oval in diameter of 100～200nm under transmission electron microscope.The interior of the particles was a dense electron core,surrounded by a envelope with spikes on the membrane.(2)NiV F and G proteins were expressed by prokaryotic expression vector pET30a,and were purified by Ni2+ affinity chromatography.F and G proteins were subcutaneously immunized to rabbits to obtain immune sera.The rabbit serum antibody titers of the two proteins reached 1:40960 detected by indirect ELISA.(3)The serum neutralization test showed that NiV pseudovirus had neutralization effect with rabbit serum immunized with F or G protein,which proved that the NiV pseudo virus constructed in this study could be used in serum antibody detection and titer assessment3.Expression and identification of NiV nucleoprotein with baculovirus expression system(1)NiV nucleoprotein was expressed and purified by baculovirus expression system(pFastBacHT A vector)and Ni2+affinity chromatography.SDS-PAGE detection showed that the purity of recombinant protein rBac-N was more than 90%(2)The recombinant baculovirus rBac-N was identified,indirect immunofluorescence(IFA)detection showed specific green fluorescence,Western blot detection showed specific target bands.Conclusion:1.A Taqman qRT-PCR detection method of NiV was established,which has high specificity,high sensitivity and good reproducibility,and can be used for nucleic acid detection of NiV.2.NiV pseudovirus was successfully packaged and rabbit serum antibody neutralization test showed that the pseudovirus could be used for serum neutralization antibody detection.3.NiV N protein was expressed and purified,which lays the foundation for the preparation of antibody and the establishment of serological detection method for NiV antigen. |
