| Carassius auratus is the sixth most important fish in freshwater aquaculture in China,and its rich nutritional value and delicious taste are highly appreciated by consumers.With the expansion of the crucian carp farming industry,the intensive farming model has led to the exposure of disease problems.As the carp farming industry continues to expand,the intensive farming model has led to the exposure of increasing disease problems.Cyprinid herpesvirus 2(Cy HV-2)is one of the major pathogens affecting the health of crucian carp,with a high infectivity and infection rate,and an outbreak mortality rate of 90%-100% in fish.Cy HV-2 is a linear double-stranded DNA virus with a total length of about 290,304 bp,which is in the shape of a 24-sided capsid and has a lipid envelope of the viral glycoprotein.The maturation process of Cy HV-2 occurs in the Golgi apparatus,and mature virions can be regarded as Cy HV-2 virions with envelopes in cell vesicles.The virus accumulates in the liver,hematopoietic cells of the kidney,and gills of the host in large numbers Therefore,sick crucian carp usually manifests as gill necrosis and ecchymosis,and hemorrhage symptoms appear on the outer surface,gills,fin base,internal organs and other parts.The proteins play a key role in the invasion of the host by the virus,and the capsid proteins have good antigenic properties and stimulate specific immunity in the host cells.Previous work in our laboratory identified ORF66 as the nucleocapsid protein of Cy HV-2 by mass spectrometry,so the biological function of ORF66 may provide new clues and help in the study of Cy HV-2 infection mechanism.In this experiment,an ORF66 recombinant plasmid was constructed;a mouse-derived polyclonal antibody was prepared against the r ORF66 expressed protein,and the specificity and potency of this antibody were tested.The r ORF66 protein and its polyclonal antibody were used to initially assemble a Cy HV-2 antibody colloidal gold quick test strip.Details are as follows.(1)Based on the fact that the protein encoded by ORF66 is a nucleocapsid protein,the amino acids encoded by ORF66 were predicted and analyzed by bioinformatics,and the region with rich epitopes was selected for truncation,and specific primers were designed based on the truncated protein sequence Amplify the target gene,connect the product to the p ET-28 a vector after enzyme digestion,obtain the expression plasmid,and then transform it into BL21 competent cells for induction expression,and use IPTG with a working concentration of 0.5 m M as the inducer to induce overnight at low temperature,carry out the cell crushing instrument to crush the cells,and obtain the recombinant expression protein as inclusion body protein through solubility analysis;therefore,in this paper,the inclusion body was lysed by the high concentration of urea,and the protein was denatured by gradient dialysis to recover some of its biological activity and then purified by Ni column to obtain a high purity protein.Thus,the mechanism of the role of ORF66 in the process of Cy HV-2infection can be investigated.The purified r ORF66 protein was tested for concentration and then subjected to a phage display to screen for interacting peptides.After three rounds of screening,a peptide sequence with a strong affinity to r ORF66 was obtained,and the results of the online comparison showed that host Leukotriene B4 receptor 1 could interact with ORF66.(2)Mice were immunized with the high purity Cy HV-2 r ORF66 protein obtained from the previous experiments,and mouse-derived r ORF66 polyclonal antiserum was prepared from mouse eye blood after five immunizations;protein samples were prepared from Cy HV-2 infected Gi CF cells constructed and preserved in our laboratory,and the protein samples were analyzed by Western Blot for specificity and potency,and the polyclonal antiserum was used to detect changes in ORF66 during Cy HV-2 infection.The results showed that the polyclonal antibody specifically recognized the ORF66 protein and the titer is higher than 1:10 000;After Gi CF cells were infected with the Cy HV-2 virus for 72 h,the expression of ORF66 was significantly increased,and the transcription level was the same as the translation level.Therefore,the polyclonal antibody prepared in this experiment can be used as the material for the establishment of the Cy HV-2 detection method.(3)The laboratory-preserved YC01 strain was used to attack crucian carp for 72 h after the onset of disease and Cy HV-2 could be detected in the kidney tissues of the fish.The serum of the onset of disease was collected from the crucian carp and detected by Western Blot using the crucian carp Ig M monoclonal antibody(Yancheng Institute of Technology)to determine whether the serum was stimulated by the virus to produce the virus antibody immunoglobulin M(Ig M)and whether the Ig M could recognize the r ORF66 protein.The results showed that the collected sera contained Ig M and were able to bind to r ORF66.Therefore,in this experiment,based on the good antigenicity of r ORF66,sufficient rabbit-derived r ORF66 polyclonal affinity antibodies were prepared,the protein was coupled with nanogold particles to form colloidal gold protein couples as tracers,Protein A was used as the detection line(T)and the r ORF66 polyclonal antibody was used as the quality control line,and the Cy HV-2 antibody colloidal gold quick test strips were assembled and preliminary testing.The results showed that the test strips were able to detect Cy HV-2 antibodies in positive samples,but the sensitivity and specificity of the test strips need to be further optimized. |
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