| Cyprinid herpesvirus 3(Cy HV-3),also known as koi herpesvirus(KHV),is a highly infectious koi herpesvirus disease(KHVD)that causes carp,koi and its variants dead.Koi herpes virus disease is a disease identified at the end of the 20 th century,and has now spread globally.It seriously threatens the safety of carp and koi breeding industries.It is a disease that must be declared by the World Organisation for Animal Health(OIE)and is classified as a second-class disease in our country.At present,there is no medicine to prevent and control koi herpes virus disease,and vaccine is the most effective way to control the disease.Attenuated vaccines and modified live vaccines have been shown to have significant anti-disease effects in the experiments and have a certain protective effect on carp.However,one of the characteristics of herpes virus is the ability to lurk the host after the initial infection.Under certain stress conditions,the latent virus can be activated to cause host disease.Existing attenuated vaccines are at risk of regaining virulence,so safety is a concern.The protection effect of KHV inactivated whole virus vaccine is not good.After immunizing the carp with the liposome vaccine prepared by KHV inactivated virus,the immune protection rate is improved to a certain extent,but the cost is higher.Other vaccines such as inactivated vaccines,common DNA vaccines,and subunit vaccines also have disadvantages such as safety,low immune protection rate,and high cost,which are not conducive to clinical application and promotion.In this thesis,through the development of a safe,efficient,and low-cost carbon nanotube-containing nucleic acid vaccine,it was found that the vaccine can be used to immunize fish by intramuscular injection and immersion to obtain a good immune protection effect in a certain dose range,and effectively reduce the fatality rate of KHVD,which lays the foundation for the practical application of subsequent vaccines.The content of the full text is mainly divided into the following three parts:1.Construction of single-walled carbon nanotube-carried koi herpesvirus ORF149(SWCNTs-ORF149)nucleic acid vaccine: Based on data accumulated in early laboratory research and pre-experiments performed in this project,the koi herpesvirus envelope protein ORF149 was selected from candidate antigen proteins as the target protein for this experiment.A single-walled carbon nanotube(SWCNT)was used as a transport vector to construct a koi herpesvirus(KHV)ORF149 nucleic acid vaccine.First,the pc DNA3.1(+)-ORF149 recombinant plasmid was constructed,and its expression was determined by transient transfection and immunoblot analysis.Finally,the recombinant plasmid was coupled to SWCNTs by 1,3-dipolar cycloaddition reaction method.The results showed that the transfected recombinant plasmid could detect specific signals in the immunoblot analysis and indirect immunofluorescence test,but the blank control group did not respond.The recombinant vaccine constructed in nucleic acid agarose gel electrophoresis electrophoretic traces disappeared;under field emission scanning electron microscope observations,the morphological differences between SWCNTs coupled with the recombinant plasmid and blank SWCNTs were obvious.The single-walled carbon nanotube-loaded KHV ORF149 new nucleic acid vaccine was successfully constructed,laying a foundation for subsequent research on immune effects.2.SWCNTs-ORF149 nucleic acid vaccine via intramuscular injection immunization and effect evaluation: we divided healthy koi(15 ± 2cm)into 9 groups,each with 60 tails,and with the recombinant nucleic acid vaccine(SWCNTs-ORF149)constructed in the previous step,three groups were immunized at three doses of 1 μg,5 μg and 10 μg via intramusclar injection,and the remaining six groups were set as control groups.Recombinant plasmid expression testing in vivo after injection showed that ORF149 successfully entered the fish body and expressed;serum titer testing 1 to 8 weeks after immunization found that serum antibody levels in the three experimental groups were significantly higher than those in the blank control group,of which 10 μg injection dose group was the highest and reached its peak value at the 4th week.The detection of immune-related genes showed that the immune-related genes in the experimental group were all up-regulated.The 10 μg group had the largest up-regulation,which was consistent with the serum test results.The results of acid phosphatase(ACP),alkaline phosphatase(AKP),and superoxide dismutase(SOD)activities,etc.,showed that the experimental group significantly increased,the 10 μg group was the highest,consistent with the above results.Finally,the challenge test was performed 4 weeks after immunization.The results showed that the mortality rate in the experimental group was lower than that in the blank control group.The mortality rate in the 10 μg group was the lowest among the nine groups,and its relative immune protection rate reached 81.9%,which was significantly higher than the other groups.It shows that the recombinant nucleic acid vaccine(SWCNTs-ORF149)can effectively reduce the lethality of KHV and achieve good immune protection effect by immunization with intramuscular injection.3.Immunization and effect evaluation of SWCNTs-ORF149 nucleic acid vaccine via immersion: we divide healthy koi(3 ± 2cm)into 9 groups,60 in each group,and immerse the recombinant nucleic acid vaccine(SWCNTs-ORF149)by immersion,respectively.Three groups were immunized at three doses of 1 mg/L,5 mg/L,and 10 mg/L,and the remaining six groups were set as control groups.Serum titer test at 1-8 weeks after immunization found that serum antibody levels in the 10 mg/L experimental groups were significantly higher than those in the blank control group,and the antibody level in the 10 mg/L injection dose group was the highest and reached peak value at 4th week;immune-related gene testing showed that both high-dose experimental groups had immune-related genes up-regulated,and the 10 mg/L group had the largest increase,consistent with serum test results;The results of detection of immune-related physiological indicators,such as acid phosphatase(ACP),alkaline AKP,SOD activity,etc.showed that both the 10 mg/L experimental groups significantly increased,the 10 mg/L group was the highest,consistent with the above results;finally,a challenge experiment was performed after 4 weeks from the immunation,and the results showed that the mortality rate in the experimental group was lower than that in the blank control group.The mortality rate in the 10 mg/L group was the lowest among the nine groups,and its relative immune protection rate reached 56%,which was significantly higher than the other groups.It shows that recombinant nucleic acid vaccine(SWCNTs-ORF149)can also effectively reduce the lethality of KHV by immersion immunization.In this paper,by constructing a single-walled carbon nanotube-coupled koi herpesvirus ORF149 recombinant nucleic acid vaccine,it is studied that this new type of nucleic acid vaccine can produce better immunity against koi herpes disease under the action of two types of immunization methods,which will lay the foundation for the subsequent practical application of vaccines. |
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