Preparation Of DNA Vaccine And Yolk Antibody Of CyHV-2 And Detection Of CyHV-2 In Fish Eggs

Cyprinid herpesvirus II?CyHV-2?is a highly pathogenic DNA virus.In recent years,the disease has spread out large-scalely in the Yancheng area of Jiangsu Province,it has Shows high lethality in Alpaca,which has caused huge losses to the aquaculture industry.At present,the inactivated vaccines and attenuated vaccines for preventing and treating the disease are generally ineffective and have poor safety.Therefore,it is urgent to develop a safe and effective new vaccine for the effective prevention and control of CyHV-2.1.In this study,the baculovirus surface-displayed vaccines of ORF72 and ORF66 of Cyprinid herpesvirus II and the nucleic acid vaccines of D4 ORF of Cyprinid herpesvirus II were constructed,And evaluating their immune effect of anti-CyHV-2.The ORF72 gene and ORF66 gene of CyHV-2 were cloned into the pFastBacTMDual-P10-GP64 CTD vector of baculovirus surface display.Wo obtained the two recombinant viruses through the system of Bac to Bac,the two recombinant viruses were named as BmNPV-P10-GP64-ORF72 and BmNPV-P10-GP64-ORF66.Which infected BmN cells respectively.After 3 days of the infection,the viral particles were collected by ultracentrifugation,and determinded the titer of two recombinant viruses.Two recombinant viruses were injected with amount of 1.0×10¹⁰TU per tail fish?the body length is 7-13 cm,the body weight is 35-60 g?,and control group were injected with equal volume of normal saline.After 3 weeks,the level of antibody titer in the blood was measured by the assay of competitive enzyme-linked immunosorbent?ELISA?.The results showed that there was no significant difference in antibody titers between the immunized group?2 groups?and the control group.It showed that the two surface-displayed vaccine was not effective against herpes virus type II.The capsid protein?ORF66,ORF72?of CyHV-2?Gene bank:JQ815364?and the vesicular protein?ORF81,ORF82?of CyHV-2 were selected as candidate vaccine proteins.which were predicted as antigen epitope mapping.Their DNA sequence were cloned into transfer vectors of recombinant baculovirus,which was controled with?-actin promoter,and the recombinant virus is constructed through the system of Bac-to-Bac,which was named as BmNPV-FA-D4 ORFs.Wo Immuned fish by the ways of injection and oral administration of?the body length is 7-13 cm,the body weight is 35-60 g?.The results showed that the BmNPV-FA-D4 ORF was vaccinated by intraperitoneal injection of cyanobacteria?2.2×1013 TU/tail viral load?,and BMNPV was used as a negative control and blank control group?normal volume saline/tail?.The DNA and RNA levels of the relevant vaccine genes in the immune fish body were detected by PCR and rt-pcr in the first,second and third week of the fish immunization.Immunohistochemical methods were used to detect the expression of vaccine gene protein levels in the spleen and kidney of the immune system.And the level of antibody titer was detected by competitive-ELISA at the 1st,2nd,3rd,and 4th weeks after immunization;the homogenate of CyHV-2 was infected at the 3rd week after immunization,and the immune effect was evaluated.The results showed that the BmNPV-FA-D4 ORF vaccine was able to enter the fish tissue successfully after being immunized with fish,the antibody was produced in the immunized group and reached the highest level in the third week.The mortality rates of the immunization group,the negative control group,and the blank control group after the challenge were 7.69%,29.17%,and 38.46%respectively.Virus drops to 2.2 x 1011 TU in ul/mg/ml of vaccine in granular feed is added to the proportion of fish feed?control group was added into an equal volume of physiological saline?feeding allogynogenetic crucian carp and infecting CyHV-2disease tissue of slurry after 4 weeks.The death rate of the immune group and the blank control group was 15.38%and 25.93%respectively.The results showed that the BmNPV-FA-D4 ORF was established by injection and oral administration,which had a good immune protection effect on the gills.The crucian carp was immunized by immersing the immunized 500 copies of the 1 ul vaccine of water.After 5h,the immune group gills,spleen and blood were taken,and the vaccine gene of them was detected by rt-pcr.The results showed that the vaccine related genes were detected in the spleen,spleen and blood of the immune group,indicating that the BmNPV-FA-D4 ORF vaccine could be expressed in the fish by soaking.2.Preparation of the yolk antibody of ORF72 protein of the herpesvirus II virus.In order to investigate the protective effect of yolk antibody against herpes simplex virus II.the CyHV-2 ORF72 gene was cloned into PET28+?a?and expressed in E.coli.The results of SDS-PAGE and Western-blot showed that ORF72 was successfully expressed in E.coli with high expression level.In the fermenter,the bacteria were collected after amplification and culture,and the soluble ORF72 protein was purified from the transformed E.coli through a series of processes such as disruption,denaturation,refolding,Ni column,elution,and dialysis.By immunizing chickens,eggs were collected to prepare egg powder andabout 100 kg of egg powder was obtained totally.Western-blot test results showed that egg powder contains specific ORF72 antibody,ELISA test results show that the antibody titer in egg powder is 1;8,000-16,000.These lay the foundation for testing the effect of yolk antibodies in the prevention and control of hemorrhagic diseases.3.Detection of CyHV-2 in the fish eggs of carassius auratus gibelio.We designed primers based on the helicase gene of CyHV-2 virus and established a set of real-time PCR detection methods to detect the infection differences in different tissues of carassius auratus gibelio.The test results showed that there was a significant difference in the degree of infection of the heart,liver,spleen,kidney,and sputum between the onset of crucian carp.The degree of infection of the kidney was the highest,followed by spleen and sputum,and the level of infection was lowest in the heart.At the same time,the toxicity difference of cyhv-2 in the eggs of the fish with no symptoms was detected:the fish eggs with no symptoms were 2.5 percent.The toxic rate of the fish eggs was 12.5 percent,and the toxic amount was 3.31 copies of the fish eggs.The two results also showed a weak positive correlation between CyHV-2 levels of heterotrophic silver cockroach and the amount of virus in eggs.These evidences indicate that there may be a vertical transmission pathway of CyHV-2 virus between the parents and offsprings of the crucian carp.