| Duck viral hepatitis(DVH)is an acute,highly lethal,contact infectious disease caused by duck hepatitis virus(DHV).The liver is swollen with bleeding points,splenomegaly,angulation,and unstable walking.At present,the popular DHV in China is mainly type 1 duck hepatitis A virus(DHAV-1),which is also the most popular serotype in the world.The DVH caused by the virus caused huge economic losses to the farmers,which seriously restricted the healthy development of the duck industry.Due to the acute onset of the disease in clinical production,the mortality rate is high,and the laboratory test method is troublesome and time consuming,which is not conducive to the rapid diagnosis and treatment of the disease.In order to facilitate the rapid diagnosis of the disease in clinical production,this study developed a colloidal gold test strip for rapid detection of DHAV-1.This study is divided into three parts.Experiment 1.Induction and expression of recombinant VP1 protein of DHAV-1: The recombinant plasmid pET32a-VP1 recombinantly expressing DHAV-1 VP1 protein was re-identified and sequenced,and the recombinant protein was induced and expressed.Blotting identification analysis.The results showed that the VP1 protein of DHAV-1 was successfully expressed,and the purified protein was purified at a concentration of 2 mg/mL.Western blotting analysis showed that the recombinant protein could react with DHAV-1 monoclonal antibody 4E6 and had a good reaction.Experiment 2.Preparation of polyclonal antibody and monoclonal antibody to DHAV-1:the recombinant protein was inducing expressed,purified,and injected into BALB/c mouse with the dose of 100 ?g per mouse,and immunized once every two weeks.At the 5th intraperitoneal injection,the titer of the polyclonal antibody was detected by ELISA.The results showed that the titer of polyclonal antibodies increased rapidly in the early stage,and gradually stabilized in the later stage.The titer of the polyclonal antibody was 1:25600.The hybridoma cell line 4E6 secreting DHAV-1 monoclonal antibody was resuscitated,and injected into paraffin-embedded BALB/c mice 7 days earlier,and 10 days later,ascites wasinjected into the mice,and the titer of the monoclonal antibody was detected by ELISA.The ELISA test result showed that the titer of monoclonal antibody 4E6 was 1:51200.Experiment 3.Development of DHAV-1 colloidal gold test strips: the colloidal gold particles of different diameters were prepared by trisodium citrate reduction method,and the colloidal gold particles and purified antibodies were produced after adjusting to the optimum pH value.Physically bound to the glass cellulose membrane;the purified monoclonal antibody was used as a capture antibody,immobilized on a nitrocellulose membrane,and a colloidal gold immunochromatographic test strip was prepared,and the optimal working conditions were optimized.The optimal concentration of the colloidal gold-labeled antibody was determined to be 15 ?g/mL,and the optimal labeling value was 18 ?g/mL.The T-line concentration of the coating on the NC membrane was determined to be 700 ?g/mL.The coating C line concentration was 600 ?g/mL.The specificity,sensitivity,reproducibility and stability of the prepared colloidal gold test strips for DHAV-1 were tested.The results showed that the prepared colloidal gold test strips were reacted with DHAV-1 and DHAV-3 not cross-reacted with other duck viruses.The sensitivity test showed that the prepared colloidal gold test strips could detect the minimum viral protein content being 9.198 ng/?L,and the sensitivity was slightly lower than the double antibody sandwich method.After stored at 4 ° C and 25 ° C for 6 months,the test results of the strips did not be affected.This indicated that the prepared colloidal gold test strips for DHAV-1 detection in this study has good specificity,sensitivity,reproducibility and stability,which provided a rapid,clinical diagnosis for DHAV-1 for providing test results within 5 min. |
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