| Duck viral hepatitis is a kind of highly contagious and acute infectious disease of young ducklings mainly within the 3-week-old ducklings, which is caused by duck hepatitis virus A. The mortality of this disease can be 90%. VP1 protein exposes to the most external of DHAV. and it is the major structural protein of DHAV and acts as the main determinant of DHAV’s antigenicity. It is reported that there is cross reaction between DHAV-1 and DHAV-3. In order to establish a simple and fast assay to detect antibodies against DHAV-1 and DHAV-3. we optimize the codons of VP1 of DHAV-1 X strain to obtain optiVPl. and it was subcloned to pET-28a(+) to obtain structural protein optiVPl in our study. Besides, we established a rapid and convenient immunochromatographic strip test based on the purified optiVPl protein for the detection of DHAV-1 and DHAV-3 specific antibodies. There are four parts in this thesis.1. Rare codons and antigenic epitopes analysis of VP1 of DHAVWe analyzed the rare codons and antigen epitopes of VP1 of DHAV-1 X strain by bioinformatics analysis online websites. We found that there were 26 rare codons and 5 consecutive rare codons. The sequence of 1-72AA and 83-238AA were full of antigen epitopes. Besides, we optimized VP1 and artificially synthesized optiVP1 according to the codon bisas of E.coli.2. Expression, purification and identification of VP1 and optiVPlExpected sized VP1 was obtained from RT-PCR. and it was successfully cloned to pMD19-T-simple. Then VP1 and optiVPl were correctly cloned into pET-28a (+) vector, respectively. The recombinant plasmid pET-28a (+)/VP1 and pET-28a (+)/optiVP1 were expressed in E.coli. However, only the optiVP1 was expressed successfully with a molecular weight about 32 kDa in BL21. And it was in the form of inclusion bodies anyway. Besides, the expression of optiVPl protein could reach top when induced by 0.4 mmol/L IPTG overnight at 37℃. The purification by using gel extraction proved high pure. Western blot analysis indicated the recombinant protein was able to react with antibodies against DHAV-1 and DHAV-3. which showed good antigenicity.3. Preparation of optiVPl antiserumsThe serum of mouse-anti-optiVP1 was prepared by immunizing the mouse with purified optiVPl protein. The antibody titer of the serum was 1:51200 by indirect ELISA. And the serum could recognize DHAV-1 and DHAV-3 successfully through indirect immunofluorescence technique, which suggested good immunogenicity.4. Development and application of an immunochromatographic strip for detecting antibodies against DHAVTwo different immunochromatographic strips (ICS), ICS-SM and ICS-ID based on optiVPl protein were developed. Both of these two ICS could detect the antibodies against DHAV-1 and DHAV-3, and the results of the detection for antibodies against Riemerella anatipestifer, Duck plague virus, Duck Escherichia coli, Duck salmonella enteritidis were negative, which meant the specificity of these two ICS were good. The detection result of DHAV-1 and DHAV-3 antiserum were positive when the dilution rate were 1:64 and 1:32, respectively. The sensitivity of the ICS-SM was as good as or even higher than neutralization test, and that of ICS-ID was as good as neutralization test. Coincidence rate of these two ICS between indirect ELISA were 82.3%(93/113) and 85.8%(97/113), respectively, and the coincidence rate between neutralization test were both 100%(16/16). What’s more, the stability of these two kinds of ICS were good, and they could be stored at 4℃ for at least 6 months. |
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