| Duck parvovirus can cause duck beak atrophy and dwarfism syndrome.Diseased ducks are characterized by atrophy and deformation of beak,protruding tongue and growth retardation.At the end of 2014,the disease occurred and reported for the first time in China,which caused serious losses to the meat duck breeding industry in China.At present,there is no commercial antibody detection kit and yolk antibody preparation for duck parvovirus.Based on this,this study intends to establish an ELISA method for detection of duck parvovirus antibodies and prepare refined egg yolk antibodies against duck parvovirus.Firstly,the VP2 gene of duck parvovirus was amplified by specific primers and inserted into pET-3 2a(+)vector to construct prokaryotic expression system,and optimize the conditions for expression.The results showed that the recombinant VP2 protein was located in the inclusion body with a size of 49 kDa,and the optimal concentration of IPTG induction was 1.0 mmol/L.Western-bloting results showed that the recombinant protein was specific to antibody serum.Then,the antibody ELISA detection method was established and tested with this protein.The results show that the ELISA method established in this experiment has good specificity,sensitivity and reproducibility,and can be used to detect duck parvovirus antibodies.Secondly,duck parvovirus vaccine was used to immunize laying hens,and yolk antibodies were prepared.Duck parvovirus yolk antibodies were purified by three different methods and routine tests were carried out.The results showed that the EID50 of the vaccine was 10-4.84/0.2 mL,and the dosage form,viscosity,stability and safety of the vaccine were all qualified.The routine test results of yolk antibody showed that the purification effect of water dilution-ammonium sulfate secondary precipitation method was better.The yolk antibodies prepared by the three methods were relatively pure.The concentrations of yolk antibodies prepared by water dilution-ammonium sulfate secondary precipitation method,caprylic acid method and chloroform method were 3.837 mg/ml,3.422 mg/ml and 4.512 mg/ml,respectively.the color and transparency were clear and transparent,light white translucent and light yellow transparent respectively.After that,the prepared egg yolk antibody was identified by SDS-PAGE andWestern-bloting.The results showed that the egg yolk antibody had two chains,the size of 65 kDa and 33 kDa respectively,which could specifically bind to the VP3 protein of duck parvovirus and had good reactivity.The titer of prepared yolk antibody was determined by the previously established antibody ELISA detection method.The results showed that the titer of yolk antibody prepared by water dilution-ammonium sulfate secondary precipitation method was the highest,which was 1:12800.While the titer of caprylic acid method and chloroform method was 1:6400,which was slightly lower than that of caprylic acid method and chloroform method.Finally,the protection rate of egg yolk antibody was determined by challenge protection test.The results showed that the infection rate in the attack group was 100%,and the growth of beak length and body weight were obviously hindered,which was in line with the characteristics of the disease.The infection rate in the protection group was 10%.The protective rate of egg yolk antibody to susceptible ducks reached 90%,which could effectively prevent duck parvovirus infection.In this study,the ELISA method for specific detection of duck parvovirus antibody was established,and the egg yolk antibody of duck parvovirus was prepared,and the effect of egg yolk antibody was evaluated.The results of this study provide an effective means for rapid detection and scientific prevention of duck parvovirus infection,and are of great significance for the large-scale preparation and application of egg yolk antibody. |
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