| Objectives:This study was to optimize the expressed condition of recombinant stain E.coli BL21(DE3)/pET32a-L99S of S gene of SEO Hantavirus strain L99, purify the recombinant nucleocapsid protein (rNP) and prepare antisera against purified rNP, and then establish sensitive and specific immunologic assays for detecting antibodies in sera of patients with HFRS and detecting Hantavirus antigen in murine lungs.Methods:1 The expressed condition of rNP of E.coli BL21(DE3)/pET32a-L99S was optimized by observing the influence in 18 conditions of 3 factors: temperature, time and concentration of inducer. The concentration of rNP and percentage in total bacterial protein were analyzed by SDS-PAGE.2 After ultrasonication of E.coli BL21(DE3)/pET32a-L99S, the inclusion bodies were washed and for the procedure of solubilization, and then the solution of inclusion bodies were purified by Ni-affinity chromatography. After purification, the target protein was renatured through dialysis and analyzed by SDS-PAGE. The concentration of recombinant protein was measured by Bradford method and the antigenicity was identified by western blot.3 Polyclonal antisera were prepared by immunizing rabbits with rNP. The tite was detecded by ELISA and immunogenicity was identified by western blot.4 Purified rNP was applied as antigen for three different ELISAs: rNP-ELISA, HRP-rNP-ELISA and IMMS-rNP-ELISA detecting specific IgG antibody in sera of patients. Their sensitivity and specificity were detected using positive sera of HFRS and health adult sera.5 Antibodies against rNP were employed as primary antibody to detect Hantaviruses antigen in murine lung through indirect irnmunofluorescent antibody technique (IFA), and compared with IFA using mixed patient sera. The inconsistent samples in the two IFAs were confirmed by RT-nested-PCR.Results: 1 E.coli BL21(DE3)/pET32a-L99S obtained the best expression under the conditions of 0.5 mmol/L of IPTG, 30℃of temperature and 5 hours of inducing time. The result showed the concentration of rNP was 626.04μg per milliliter culture solution and accounted for 52.2% in the total bacterial protein. The targed protein exsited in the form of inclusion bodies.2 After Ni-affinity chromatography, the purity of rNP was up to 95%, concentration was 18.01 mg/ml, the overall recovery was 28.9%, the purify multiple was 1.83, and western blot showed the purified protein could be identified specifically by IgG antibody in serum of patients with HFRS.3 The tites of polyclonal antisera against rNP were 512 000。Western blot showed a single band.4 Using the three ELISAs to detecting specific IgG antibody in sera of patient with HFRS, the sensitivity of IMMS-rNP-ELISA, rNP-ELISA and HRP-rNP-ELISA were 100%, 100% and 92.5% respectively, and the specificity were 100%, 95% and 100% respectively.5 59 murine lung samples were detected for Hantavirus antigen by two kinds of IFA. The positive detection rate was 23.7% using antisera against rNP and 16.9% using mixed patient sera. The background of sections using antisera against rNP were clearer than sections using mixed patient sera. RT-nested-PCR showed all the inconsistent samples in the two IFAs corresponded to the results of IFA using antisera against rNP.Conclusion:1 The expressed and purified condition of recombinant stain of E.coli BL21(DE3)/pET32a-L99S were determined.2 RNP was applied to rNP-ELISA, HRP-rNP-ELISA and IMMS-rNP-ELISA for detecting specific IgG antibody in serum of patients with HFRS, the methods were sensitive, specific, simple and easy to spread.5 We prepared antisera against rNP and using them to monitor sensitive animal, and the method was worth adopting widely. |
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