Immunogenicity Of Glycoprotein Gn Of Severe Fever With Thrombocytopenia Syndrome Virus

Objective:To study the humoral and cellular immunogeniciry of glycoprotein Gn of severe fever with thrombocytopenia syndrome virus.Methods:1. According to the glycoprotein Gn coding sequence of SFTSV HB29 (GenBank: HM745932.1) and optimize its codons with the amino acid sequence unchanged. The wild type sequence WSP-Gn was synthesized, then the introduction of restriction enzyme sites HindⅢand BamH I was linked the WSP-Gn or Nhe I and BamH I was linked the Gn by PCR. The above gene product were cloned to pJW4303 to construct recombinant plasmids pJW4303-WSP-Gn and pJW4303-tPA-Gn. The optimized sequence WSP-Gn-opt was synthesized, then the introduction of restriction enzyme sites Nhe I andBamH I was linked the Gn-opt by PCR. The gene product was cloned to pJW4303to construct recombinant plasmids pJW4303-tPA-Gn-opt.2. Western blot was used to detect the expression of glycoprotein Gn in the super-nates and lysates harvesting from 293T cells after transient transfection with reco-mbinant plasmids, and pJW4303 as a control.3. We immuned the BALB/c mice with recombinant plasmids and blank vector at 0,2, 4.8 week. Blood were collected prior to the each immunization and 6,10 week after the first immunization, and sera specific IgG, IgG1, IgG2a antibodies were detected by ELISA.4. Predicting the CTL epitopes of Gn by bioinformation and 16 peptides (P1-P16) were choosed to carry out follow-up experiment. Meanwhile, BALB/c mice were immunized with recombinant plasmids and blank vector at 0,2,4 week. Mice spleen cells were analyzed to determine the IFN-y positive lymphocyte by Elispot at 17,31, 60,90 day after the first immunization.Results:1. The pJW4303-WSP-Gn, pJW4303-tPA-Gn and pJW4303-tPA-Gn-opt were correc-tly cloned and identified by restriction enzyme digestion and sequencing.2. The expression of glycoprotein Gn in 293T cells:The pJW4303-WSP-Gn and pJW4303-tPA-Gn transiently expressed Gn antigen in cell lysates of HEK293T cells in vitro, and pJW4303-tPA-Gn-opt in supernatants as same as cell lysates.3. Humoral immune response:pJW4303-WSP-Gn, pJW4303-tPA-Gn and pJW4303-tPA-Gn-opt could induce the specific anti-Gn antibodies IgG, IgG1 and IgG2a in the serum after immunization in mice. The specificantibodies induced by pJW4303-tPA-Gn-opt was earlier and higher than that of pJW4303-WSP-Gn and pJW4303-tPA-Gn. The IgG2a/IgGl ratio of pJW4303-tPA-Gn-opt induced was approximatey 1, which resulted in most balanced Th immune responses in mice.4. CTL immune response:The peptides P1 and P10 could stimulate aplenocytes secreting IFN-y after immunization of recombinant plasmids, and the P10 was superior to P1. The CTL immune response of pJW4303-WSP-Gn group stimulated by P1 was better than the others, and pJW4303-tPA-Gn-opt group stimulated by P10 was better than the other gropus.Conclusions:1. Glycoprotein Gn of SFTSV had expected humoral and cellular immunogenicity2. P1、P10 are the CTL epitopes of glycoprotein Gn of SFTSV.