Recombinant Expression Of Hantaan Virus Protein N And Establishment And Application Of RNP-IgM Direct Capture ELISA

Hemorrhagic fever with renal syndrome (HFRS), which is cuase by hantavirus, is an acute infectious disease characterized by fever, shock, hemorrhage and kidney dysfunction. hantavirus (HV) is a worldwide viral infectious disease and China is a main epidemic area. An estimated 40,000-60,000 cases occur annually.HV belongs to the family Bunyaviridae, and the genome is a single-stranded negative-sense RNA including a tripartite genome, large (L) gene, medium (M) gene and small (S) gene coding for the viral ploymerase, two envelope glycoprotein (G1 and G2) and nucleocapsid protein (NP), respectively.Though RT-PCR could be used for the diagnosis of HFRS, a proportion of acute cases is difficult to be diagnosed because of the short period of persistence of HV in the body with a low viral titer. Therefore, the serological diagnose of the specific antibody will be served as a reliable test for HFRS.During the acute phase of infection, the immunoglobulin M (IgM) level rises, followed by the production of IgG; the early antibody response is induced by nucleoprotein N, the major antigen. Serological assays are based on viral antigens expressed in infected cells. However, massive production of viral proteins is rarely observed because the virus grows poorly in tissue culture. In addition, some hantaviruses must be manipulated in a high-security containment facility. Therefore, several laboratories have expressed the N protein as a recombinant protein in Escherichia coli or in insect cells. Thus, NP can beregarded as an essential antigen for the HFRS serological diagnosis.Many authors abroad have established various expression systems to express NP used as the diagnostic antigen for HFRS serological test.However, the present HFRS serological test, which needs the viral antigen and 4 main operation steps, is an elaborate and time-consuming procedure. So the establishment of a new rapid, simple, safe, sensitive and specific HFRS serological test is of great importance in the diagnosis of HFRS.In this study, we cloned S-gene coding region for the establishment of NP prokaryotic expression system pET28a-Z10N-E.coli BL21DE3 with HV-Z10 virus strain genome, which had been used for the manufacture of HFRS inactivated vaccine and detecting protein expression by ELISA. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immunoreactivity of rNP. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HRP-labeled recombinant NP (rNP) is to establish IgM direct capture ELISA. Ninety-five specimens of HFRS patient's serum were tested for the comparison of the testing results with that of the routine IgM indirect capture ELISA using HV as the antigen. 94.73% (90/95) of HFRS pateints' serum samples are positive confirmed by rNP-IgM direct capture ELISA, while the positive rate in the same samples confirmed by HV-IgM in direct capture ELISA was 92.63% (88/95). The distributions of OD₄₅₀ values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the OD₄₅₀ mean values from several serum samples with different dilutions were similar. Conclusion We successfully construct a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study can be used as a new serological test for HFRS diagnosis because of its simplicity, safety, sensitivity and specificity.