| Crimean-Congo hemorrhagic fever is a tick-borne zoonotic disease caused by Crimean-Congo hemorrhagic fever virus.Vertebrate animals such as ruminants,rodents and migratory birds are susceptible.Humans are infected with severe viral hemorrhagic fever,with a fatality rate of up to 30%.CCHFV is mainly transmitted through close contact with blood,skin and mucous membranes,and there are no officially approved vaccines for animal or human use.CCHF has been prevalent in countries in Africa,the Balkans,the Middle East and Asia.CCHFV is a negative-strand RNA virus,and the genome consists of S,M,and L fragments.The envelope spike glycoproteins Gn and Gc are encoded by M gene.CCHFV glycoproteins contain linear B cell epitopes and abundant antigenic epitopes,which are important targets for CCHF vaccine development.In this study,based on the structural proteins Gn and Gc,the extracellular domain Gn1(aa525-701)of Gn and the neutralizing antibody epitope region Gc1(aa1148-1571)of Gc were expressed and purified by Escherichia coli prokaryotic expression system.Two indirect ELISA methods for detecting CCHFV antibodies were established using purified recombinant proteins Gn1 or Gc1 as coated antigens.The method was used to evaluate the immune effect of CCHFV-BLPs vaccine prepared in our laboratory.The following studies were mainly carried out:1.Prokaryotic expression and purification of CCHFV glycoprotein.Referring to the full length of the M gene sequence of CCHFV published by NCBI,the Gn1 and Gc1 gene fragments were obtained through PCR,which were 534 bp and 372 bp respectively.They were cloned into the p ET-30a(+)vector.The p ET-30a(+)-CCHFV-Gn1,p ET-30a(+)-CCHFV-Gc1 recombinant plasmids were constructed.The recombinant plasmid were transformed into DE3(BL21)competent state and expressed by IPTG induction.The glycoprotein Gn1 and Gc1 were obtained with molecular weights of 35 k Da and 23 k Da,respectively.The optimal expression conditions were optimized: the induction temperature was37℃,the concentration of inducer was 0.4 mmol/L,the induction time of Gn1 was 7 h,and the induction time of Gc1 was 3 h.Finally,both proteins are expressed in the form of containing body,and the purity of protein is more than 90%,which lays the laboratory foundation for the establishment of CCHFV antibody detection method and the research of CCHF subunit vaccine in this laboratory.2.Establishment of indirect ELISA methods for detecting of CCHFV specific antibody.The recombinant glycoprotein Gn1 or Gc1 was used as coating antigen,and the sera of mice immunized with CCHFV-BLPs-Gn or CCHFV-BLPs-Gc vaccines prepared in our laboratory was used as the first antibody and the goat anti-mouse antibody labeled with HRP as the second antibody.Two indirect ELISA methods were established.The two indirect ELISA detection methods showed good specificity,sensitivity and reproducibility.It laid a foundation for the evaluation of immune efficacy of Crimean-Congo hemorrhagic fever vaccine and the epidemiological investigation of antibody detection.3.Application of indirect ELISA methods for detecting CCHFV specifc antibody in vaccine evaluation.In order to identify the immunogenicity of CCHFV recombinant glycoproteins Gn1 and Gc1.Mice polyclonal antibodies were prepared by emulsifying the recombinant glycoproteins Gn1 or Gc1 with adjuvant and separating the serum after three immunizations.IFA identification and Western blot identification were performed.The titers of specific antibodies Ig G,Ig G1,Ig G2 a and Ig G2 b in the serum of immunized mice were detected by indirect ELISA antibody detection.The results showed that recombinant glycoproteins Gn1 and Gc1 could induce different degrees of humoral immune responses in mice.At the same time,using this indirect ELISA antibody detection method to test the serum of mice immunized with CCHFV-BLPs vaccine in our laboratory,it was found that as the number of immunizations increased,specific Ig G antibodies continued to increase. |
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