| ObjectiveHemorrhagic fever with renal syndrome ( HFRS) was an infectious disease caused by virus of the genus Hantavirus in the family Bunyavirdae . The morbidity of HFRS in our country was 90% in the world and the mortality was 3% ~ 10%. Although the major clinical manifestations of the HFRS were high fever , hypo tension, bleeding and renal damage, there were lots of difficulties in the clinical diagnosis of HFRS because of their complex clinical manifestations and the rapid change of the disease. Until now, the serodiagnosis of HFRS was still the most important method in the laboratory diagnosis. However, the sensitivity of the serodiagnosis based on the reaction of the antigen and antibody could not fit the requirement of the clinical diagnosis, therefore failing to diagnosis and leading to misdiagnosis make it possible that the patients could not get the accurate diagnosis in time, which not only delayed the treatment and even caused the patients death. Thus, the high sensitive and specific laboratory diagnostic techniques were useful to auxiliary diagnose HFRS.In this study, we carried out the following studies according to the high sensitivity and high specificity of immuno-PCR in order to set up one kind of high sensitive diagnostic method of HFRS: (1) A S gene fragment encoding the whole nucleocapsid protein (NP) of 76-118 strain of Hantavirus was inserted into pro-karyote expression vector pGEX-6P-1 to construct a recombinant plasmid pGEX-6P-1-hanS, which then was transformed to E. coli BL21 (DE3) strain for recombinant NP (rNP) expression. The antigenicity of the rNP was detected with the positive sera from the HFRS patients by ELISA and IFAT. (2) We developed immuno-PCR method, which utilized an avidin bridge to link a biotinylated antibody to a biotinylated reporter DNA, applied it to detect the specific anti-body in the sera of HFRS patients and compared with the traditional ELISA and IFAT methods. (3) The anti-HFRS-IgM antibody in the sera of the patients in the different stages of HFRS was tested by immuno-PCR method to establish one kind of early diagnostic method which was suitable to base medical units.Methods1. Construction and expression of pGEX-6P-1-hanSThe plasmid pAC-hanS was used as template for PCR amplification. The PCR product was run 1% agarose gel electrophoresis and the correct-sized band was purified and digested with Bgl fl and Xho I ; At the same time, the plasmid pGEX-6P-l -hanS was digested with BamH I and Xho I . Ligation was carried out at 16C for 45min. The recombinant plasmid was transformed into competent E. coliBL-21 (DE3) , then selected and extracted recombinant plasmids to confirm the orientation of the insert with PCR amplification and restriction enzymes.2. The expression and purification of the recombinant proteinE. coliBL-21(DE3) , which contained the pGEX-6P-l-hanS, was cultivated in Luria-Bertani ( LB) medium supplemented with 50mg of ampicillin per ml. Cultures were induced to produce recombinant protein by the addition of ImM isopropyl-p-thiogalactopyranoside (IPTG). After 5h at 37C , the culture was centrifuged and the bacterial cells were resuspended in PBS and lysed by ul-trasonication. After clarification at 10,000rpm, the supernatant containing the soluble recombinant protein was applied to a Glutathione Sepharose 4 B column, and the purified recombinant protein was detected for antigenicity with Western-Blot test.3. The detection of specific antibody in sera of patients with HFRS with indirect ELISA method.The 96-well plates were coated with recombinant antigens and blocked by blocking buffer. Subsequently, the sera of the patients were added and incubated at 37C for 30min. Finally, peroxidaseconjugated goat anti-human IgG,suffers fluids and 2M H2SO4 in sequence were added and absorbance at 490 nm was measured with a microplate reader.4. IFAT test.One hundred microliters of tenfold dilution of serum were dropped to the plate and hatched at 37C for 30min. After washed with PBS, the plate were incubated with fluore...
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