| Tick-borne encephalitis is a member of arthropod-borne acute central nervous syste m communicable diseases,which belongs to B category infectious disease in China. TBE Vs are mainly transmitted by ticks bites, which cause severe headache and fever, accom panied with respiratory failure, disturbance of consciousness, muscle paralysis and other neurological symptoms, with the fatality rate as high as5-20%. There would still be se quelae of varying severity even after the recovery. Forest areas in Northeast and Xinjian g, China, are the high incidence areas of Tick-borne encephalitis (TBE).Tick-borne encephalitis virus is a single-strand RNA virus with a capsule structure which is the pathogen of Tick-borne encephalitis, belongs to Flaviviridae Flavivirus.This virus has high genetic relationship with Japanese encephalitis virus and West nile virus.T he pathogenic mechanism of Tick-borne encephalitis virus is not clear,so there was absen t of effective therapeutic method.Nowadays the mainly TBE diagnosis method have:normal PCR?real-time PCR?E LISA?IFA?egg inoculation?mouse intracellular inoculation and plaque-forming assay.Th ese methods were very maturated in diagnosis of TBE, but aslo still some practical diffi culties.First, Virus isolation detection cycle too long,to meet the demand of the rapid dia gnosis.Second,there were some parts of conservative epitopes between Tick-borne encepha litis virus and other insect-borne flavivirus, serologic diagnosis is prone to cross-reaction may result in misdiagnosis.Third, Third, the acute tick-borne encephalitis infection, wind ow phase of viremia is very short, it was hardly success to use nucleic acid amplificatio n method detected the virus. Furthermore these diagnostic methods needs more technolog y and experience, the economy and convenience is also one of the practical factors affe ct the diagnostic results. IgM and IgG which induce by virus could maintain a longer ti me in serum, Therefore, for the detection of virus-specific antibody can improve the dia gnostic efficiency.Colloidal gold rapid detecting test is a new diagnosis technique based on colloidal gold chromotography techniques. It has a lot of advantages like rapidily?rel iability?easily and economic.In this study, we use prokaryotic express TBEV antigen pro tein to establish?optimize and evaluation the virus-specific antibody detection methods b ased on colloidal gold immunochromatography technology.At last,provide a new method f or the rapid diagnosis of tick-borne encephalitisIn this study,for ensure the specificity and sensitivity of detection of colloidal gold, prokaryotic express the Domain III from E gene and extracellular secretory NS1gene. t he primers for serial segments of Tick-borne encephalitis virus were designed according t othe MDJ01gene which isolation and identification by our laboratory recently as the te mplate DNA to amplificated E-? and NS1cDNA segments. The sub-cloned plasmids w ere constructed by connecting the reverse transcription PCR products with pMD18-T vec tor and sequenced. Then above plasmids were further digested with BamH I and Not I, and the recombinant expressing plasmids was obtained by cloning the serial segments of E-IIIand NS1genes into expression plasmid pET-30a(+). The correct plasmids identified by enzyme cleavage were transfected into competent cells of E.coli Rosseta(DE3) and t he proteins expression was induced by IPTG.. The bacteria were collected4h later at37?, and there were expected protein bands at corresponding sites in all these clones wh en analyzed by SDS-PAGE, however, all these proteins was appeared as inclusion bodies. These recombinant proteins were purified by Ni-NTA affinity chromatography, and the purity of the obtained E-??NS1proteins were above85%. Furthermore In this study, we further expressed five other segments of NS1proteins:F1R2, F1R3, F1R5, F3R2an d F2R4.The purified E-III and NS1proteins were coated onto ELISA plate to detect the T BE-positive sera of rabbit, and we found that the NS1protein and the E-? protein pos sessed the reactionogenicity, farther more the NS1protein presents more background valu e than E-III. Detection with other congeneric virus-positive sera of rabbit like:DENV?JE V?WNV. The results implied that the NS1protein and the E-? protein presented great specificity.Finally we use the recombinant E-? protein to prepared the colloidal gold detect ing test strip with the considered of all the factors.Finalize the SPA to explore optimizat ion of conditions, the pH value of7conditions mark43?g/ml amount of gold as the be st standard, testing lines and the the allegations lines crossed concentrations were determ ined to be2.5mg/ml and0.5mg/ml.And then use the anti-E-? protein monoclonal antibo dies?the TBE-positive and other congeneric virus-positive sera of rabbit detected its reac tionogenicity and specificity. Compared with IFA and ELISA, there was about94.12%c onformity in16shar clinic positive sera and18shar clinic negative sera. The res?lts of detecting the rabbit sera sample revealed that this test strip has satisfactory reactionoge nicity and detecting the clinic sera sample revealed that the test strip also has high spec ificity within5-10minutes. The test strips also possess high sensitivity thro?gh detecting the clinical samples and comparing with ELISA method. The stability was also good im plied by the2-week accelerating experiments, with a grantee period about half a year.The rapid detection method of TBEV using colloidal gold was established in this study, and this method has the advantages of good sensitivity, convenient, specificity, a nd could be further applied in clinical detection. With the establishment of this method, clinical cases could be diagnosed rapidly and remedy scheme could be determined earlie r by combining it with clinical diagnosis, epidemiological study and traditional lab detect ing methods. Thus, this method has great economic and social benefits. |
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