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Epitope Screening And Application Of GP5 Protein And M Protein Of Porcine Reproductive And Respiratory Syndrome Virus

Posted on:2022-10-09Degree:MasterType:Thesis
Country:ChinaCandidate:H LiFull Text:PDF
GTID:2480306326492744Subject:Bio-engineering
Abstract/Summary:PDF Full Text Request
As a highly contagious disease that endangers the global pig industry,porcine reproductive and respiratory syndrome has brought serious economic losses to the pig industry in China.The disease is mainly caused by porcine reproductive and respiratory syndrome virus.As the main structural protein of PRRSV,GP5 protein and M protein can induce the production of neutralizing antibodies and play an important role in the pathogenesis and immune process.The aim of this study was to screen GP5 protein and M protein epitopes,and to establish Peptide-ELISA detection methods using the selected epitope peptides,which laid a foundation for studying PRRSV antigen-antibody interaction and provided an effective detection method for the evaluation of vaccine immune effect,antibody detection and epidemiological investigation of PRRS.In this experiment,the amino acid sequences of PRRSV GP5 protein and M protein were analyzed by bioinformatics methods,and peptides were designed and synthesized according to the extracellular regions of GP5 protein and M protein.First,the extracellular region of GP5 protein(32-63 aa)and the extracellular region of M protein(1-16 aa)were synthesized.Three peptides overlapping eight amino acid were designed and synthesized according to the extracellular region of GP5 protein:GP5-1(32-47 aa),GP5-2(40-55 aa),and GP5-3(49-63 aa).The well-reacted peptides GP5-1 and M were truncated to synthesize peptides GP5-1-1(32–42 aa),GP5-1-2(38–47 aa)and M-1(1–10 aa),M-2(6–16 aa)overlapping 5 amino acids.The peptides GP5-1-2 were truncated to synthesize peptides GP5-1-2-1(38–43 aa),GP5-1-2-2(41–45 aa),and GP5-1-2-3(43–47 aa)overlapping 3 amino acids,Twelve peptides were synthesized.A cysteine was added to the N-terminus of the peptide for conjugation.(1)Dot-blot and ELISA were used to screen and identify GP5 protein and M protein epitopes;(2)Peptide-ELISA methods for the detection of PRRSV antibodies was established using the selected peptides GP5-1:32SNNSSS-HIQLIYNLTL47,M:1MGSSIDDFCNDSTAPQ16as targets conjugated with BSA,and the working conditions were optimized;(3)Marc145 cells were used as target cells to screen peptides that could block viral infection by peptide inhibition of PRRSV infection of Marc145 cells.The results showed that:(1)The epitope of GP5 protein screened by dot-blot and ELISA was GP5-1(32-47 aa),which was truncated and screened to obtain the epitope:GP5-1-2:38HIQLIYNLTL47.The epitope of M protein was M(1-16 aa),and Its screened for truncation to obtain the epitope:M-1:1MGSSIDDFCN10.(2)Through infection inhibition experiments,the extracellular domain of GP5 protein(32-63 aa)and peptide GP5-2:40QLIYNLTLCELNGTDW55could inhibit PRRSV infection of Marc145 cells at concentrations of 0.625?g/m L,1.25?g/m L,2.5?g/m L,5?g/m L,and 10?g/m L,and the effect of blocking virus-infected cells was enhanced with increasing peptide concentration.(3)Establish ELISA antibody detection method using the selected peptides GP5-1 and M,The results showed that the optimal coating amount of BSA-GP5-1 was 0.25?g/well,and the serum dilution was 1:200.The optimal coating amount of BSA-M was 0.5?g/well,and the serum dilution was 1:200.The two peptides had no cross reaction with PCV2 and CSFV swine virus positive sera,and the coefficients of variation of the intra-batch inter-batch repeatability test were less than 10%,indicating that the Peptide-ELISA method established in this experiment had good repeatability and specificity.Eighty-five clinical pig serum samples were simultaneously detected by the established peptide ELISA antibody detection method as well as the IPMA method and IDEXX kit,and the detection rates were 78.82%by IPMA method,85.88%by Peptide-ELISA method,and 95.29%by IDEXX kit.The coincidence rates between Peptide-ELISA and IPMA and IDEXX kits were 92.24%and 90.59%.In this study the Peptide-ELISA method established has higher detection rate than the IPMA method,and this method has a good application prospect in the detection and diagnosis of PRRSV.GP5 protein and M protein epitopes were successfully screened in this experiment.Safety,specific,and stable Peptide-ELISA assay were established with Epitopes of GP5 protein and M protein.Peptides that could block PRRSV virus infection of Marc145 cells were successfully screened.To provide technical support for the research and development of PRRSV antibody detection kit and provide new ideas for the prevention,control and treatment of PRRSV.
Keywords/Search Tags:Porcine reproductive and respiratory syndrome virus(PRRSV), Antigen epitope, ELISA, Antibody detection, viral blocking
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