| Crucian carp Carassius auratus gibelio has many advangtages of larger size,faster growth,more delicious meat,richer niutrition,etc.Its output continues to grow,having brought considerable economic benefits and social benefits as a result of more and more farmers breeding them.However,the large-scale outbreak of disease has hindered the development of the breeding industry of the Carassius auratus gibelio.The main diseases have parasitic diseases,bacterial diseases,fungal diseases,viral diseases and so on.Since 2012,large-scale gill hemorrhagic disease has outbreaking in carp culture area.The mortality of this disease is extremely high,causing great economic losses.The pathogen of this disease is Cyprinid herpes virus Ⅱ.The CyHV-2 was first discovered in Japanese a goldfish farm in 1992,which is named as Cyprinid herpes virus Ⅱ by the international virus system classification and naming committee because it is the second virus isolated from bream.In addition,CyHV-2 can cause carp hematopoietic tissue necrosis,thereforis also known as "Herpes viral haematopoietic necrosis virus"(HVHNV).Bream herpes virus is divided into three type:Carp pox herpesvirus(Cyprinid herpesvirus,CyHV-1);Cyprinid herpesvirus II(CyHV-2)and Koi herpesvirus(Cyprinid herpesvirus 3,CyHV-3).At present,there are many researches on CyHV-3,but there is less research on CyHV-2.There are many studies on the detection and histopathology of CyHV-2,but effective prevention and cure methods have been not found.This paper first established quantitative method for CyHV-2 in crucian carp with SYBR Green.Then the ORF72 gene was cloned to construct the prokaryotic expression vector which was used for the expression and purification of protein,and preparion corresponding antibodies.Finally,the changes of blood biochemical indicators of Carassius auratus gibelio were analyzed.These data provide valuable reference for further understanding the pathogenesis of the virus which is good for finding the effective methods of prevention and cure.The main research contents of this paper include the following three aspects:1.Establishment of a method for quantifying the CyHV-2In this experiment,the primers of CyHV-2 ORF16 gene was designed according to its gene sequences and established real time PCR method to quantify the CyHV-2 in the diseased crucian carp base on SYBR Green.The standard curve was structured firstly.Based on this data,the number of copies of virus in each tissue of diseased crucian carp were calculated.The results show that the copy number of CyHV-2 in the spleen and kidney were higher compared with the gill,liver,intestines,muscles and other tissues.These data provided a valuable information for future monitor,prevention and control of CyHV-2.2.Cloning,protein expression and antibody preparation of ORF72In this study,a pair of specific primer was designed according to ORF72 gene sequences of CyHV-2.CyHV2-ORF72 was obtained by PCR amplification with kidney DNA of disease carp as a template.The total length of the gene sequence is 1113 bp,which can encode protein of 370 amino acids.The PCR product was connected with a expression vector pET-28 to construct recombinant prokaryotic expression vector pET-ORF72,which was transformed into E.coli Transetta(DE3)and inducted recombinant protein express by IPTG.The molecular weight of recombinant protein is about 45 kDa same as we expected.New Zealand rabbits were injected subcutaneously to prepare the polyclone antibody of ORF72.The result of detection by Western blotting showed that the crude extract of the virus reacted well with the antibody.These data is helpful to establish a vaccine and immunoassay.3.The influence of CyHV-2 infection on blood biochemical indexes in Carassius auratus gibelioIn this part,the influence of CyHV-2 infection on blood physiological changes and serum enzyme activity in Carassius auratus gibelio was analyzed.In diseased fish blood,the number of white blood cells(WBC)increased significantly(p<0.05),while red blood cells(RBC),hemoglobin(HB)content,mean red cell volume(MCV),mean content of red blood cell hemoglobin(MCH),mean red blood cell hemoglobin concentration(MCHC)decreased significantly(p<0.01).The erythrocyte deposited(HCT)also showed decreased but the difference was not significant(p>0.05);Compared with healthy fish,the enzyme activity of alanine aminotransferase(ALT)、aspertate aminotransferase(AST)、alkaline phosphatase(ALP)and lactate dehydrogenase(LDH)in blood of the diseased fish was significantly increased,and the content of urea nitrogen(BUN)also increased significantly(p<0.01).In addition,the enzyme activity of total antioxidant capacity(T-AOC),acid phosphatase(ACP),GSH-S transferase(GST)and catalase(CAT)in blood serum of the diseased fish increased significantly(p<0.01).The data lays a foundation for further revealing the pathogenic mechanism of CyHV-2. |
