Development Of Monoclonal Antibody-based Detection Method For Cyprinid Herpesvirus 2 And Its Control By Traditional Chinese Medicine

Cyprinid herpesvirus 2(CyHV-2)is the causative agent of the highly pathogenic viral disease herpesvirus hematopoietic necrosis(HVHN).In recent years,the outbreak of the virus has brought a great deal of damage to the crucian carp breeding industry of China,characterized by high pathogenicity and high mortality rate.Therefore,to prevent and control the spread of CyHV-2,an accurate virus detection has been established.Methods and screening for effective prevention and control drugs are indispensable.Detection technology about CyHV-2 has been reported a lot,but there are few studies on immunological diagnostic techniques related to monoclonal antibodies in 150 genes encoded by virus,which is not conducive to early monitor and detection of viruses.Monoclonal antibody technology is based on the specificity of the antigen-antibody combination and is used to construct immunological diagnostic methods with stronger immunogenicity.So far,the research on the effectiveness of the anti-viral drugs of Allogynogenetic crucian carp is still blank.Chinese herbal medicine has a long history of human medical treatment in China.Because of its dual role of nutrition and medicinal properties,Chinese herbal medicine has attracted widespread attention.Its advantages including low cost,less drug residues and side effects,and abundant resources are also used in the prevention of diseases in aquatic animals.It is potential for using anti-virus Chinese herbal medicine as a research and prevention strategy for CyHV-2.Therefore,the study of monoclonal antibodies against CyHV-2 and the screening of anti-viral herbs are of great significance for the prevention and control of CyHV-2.The contents of the study are as follows:(1)The diseased fish tissues infected with CyHV-2 in Sheyang Jiangsu was used to extract DNA of the purified sample as a template for the PCR reaction,and amplification primers were designed based on the ORF92 gene(genome accession number: AFJ20521.1).After being cloned into prokaryotic expression vector PGEX-4T-3,it was induced to express in E.coli and the product was analyzed by SDS-PAGE.The purified ORF92 recombinant protein was used to immunize BaLB/c mice to obtain anti-ORF92 recombinant protein serum.Western blot results showed that the polyclonal antibody against the CyHV-2 ORF92 recombinant protein can be used to specifically recognize the prokaryotic expression of the ORF92 recombinant protein,but also can identify the main capsid protein ORF92 of CyHV-2 virus particles,and the use of immunohistochemistry to identify viral particles in the kidney tissue infected with CyHV-2.(2)The prepared anti-ORF92 polyclonal antibody experimental results show that the ORF92 recombinant protein can be used as an antigen to immunize mice to produce antibodies against ORF92 recombinant protein.We immunized BaLB/c mice four times with an intraperitoneal injection of the antigen.On the third day after the last immunization,the spleens of the immunized mice were fused with SP2/0 tumor cells to generate new hybridoma cells.A sub cloning of a cell line that tested positive for a monoclonal antibody capable of stably secreting anti-CyHV-2 ORF92 was named 1B7.The obtained 1B7 cell strain was expanded and cultured,cryopreserved,paraffinsensitized mice were used,and 0.5×107 1B7 cells were injected to produce a large number of monoclonal antibodies ascites.The subtype of CyHV-2-ORF92-1B7 monoclonal antibody is IGg2 b,and its antibody titer can reach 1:50000.The sections of kidney,spleen,gill,brain,muscle,heart tissues and RyuF-2 cells infected with CyHV-2 were detected.Immunofluorescence was detected using monoclonal antibody 1B7 as primary antibody.(3)By adding 1% Andrographis paniculata,Astragalus,Isatis tinctoria,or Rheum officinale to the basal feed for Carassius auratus gibelio,experimental groups were injected intraperitoneally with 1 mL of the indicated amounts of purified virus to investigate effects of Chinese herbal feed additive on Carassius auratus gibelio infected CyHV-2.After artificial infection,the titer of CyHV-2 in blood was measured by realtime fluorescence quantitative PCR.The experimental results showed that the clinical symptoms of challenged fish are hyperemia at the base of the fins and on the abdomen,gill bleeding,splenomegaly,and internal bleeding.The mortality of Carassius auratus gibelio fed with Andrographis paniculate,Astragalus,Isatis tinctoria,Rheum officinale feed was 15%,30%,35%,and 95%;while the mortality of control group reached 100%.The virus titer in the control group was higher than those in the experimental groups of Andrographis paniculata,Isatis tinctoria and Astragalus except for that of Rheum officinale.The results showed that Rheum officinale feed's effect against Andrographis paniculata is relatively poor.Compared with Astragalus and Isatis tinctoria feed's antiviral effect,Andrographis paniculata demonstrated a significant better antiviral effect against Andrographis paniculate,which indicated its potential as a natural feed additive to fight against Andrographis paniculata.The results of this study provided a theoretical basis for the prevention and treatment of CyHV-2 infection in Carassius auratus gibelio through addition of traditional Chinese medicine.