Expression And Immunogenicity Of LSDV ORF132 In Insect Baculovirus System

Lumpy skin disease(LSD)is an acute,subacute and chronic highly contagious disease of cattle caused by Lumpy skin disease virus(LSDV).The disease can cause abortion or even infertility of pregnant cows infected with primers,temporary loss of gestation function of bulls,decrease of milk yield of milk-producing cows,emaciation of infected cows,and low leather utilization rate due to nodules on the skin.The disease can infect cattle of any breed and age and cattle of any age,in which the death rate of calves can reach 100% and that of adult cattle is 3%-10%.The World Organization for Animal Health(OIE)lists it as one of the required animal diseases,and China also lists it as a Class I animal disease.In 2019,the first LSDV was found in Ili,Xinjiang..The main mode of transmission of the disease is through blood-sucking arthropod insects,which multiply and migrate in large numbers with seasonal climate changes.As international trade increases year by year,the convenience of goods transportation will also lead to the spread of the disease,because these bloodsucking insects will go to areas where the disease does not occur with transportation.Increased the probability of the disease spreading globally.Therefore,it is necessary to develop new and effective monitoring and prevention methods.At present,the main prevention and control method for the disease is vaccination with attenuated vaccine,and there is no drug treatment.Local skin adverse reactions will occur during the use of the attenuated vaccine,and the leakage of the attenuated vaccine is easy to cause environmental pollution and lead to cattle infection.In addition,due to the host specificity of heterologous attenuated vaccines,there is also a potential risk of goat and sheep infection.Not only that,the evolution of attenuated vaccine in animal body and its environment may have the risk of virulence regression becoming higher.Therefore,it is necessary to seek a new,efficient and stable vaccine to prevent and control bovine nodular dermatosis.Insect baculovirus expression system is an efficient protein expression system,and is also one of the systems commonly used to produce virus-like particle vaccines in eukaryotic cells.This study expressed LSDV specific gene ORF132 through insect baculovirus expression system,constructed recombinant baculovirus particle-like vaccine through baculovirus expression system and studied its immunogenicity,which laid the foundation for further research and development of new vaccines for bovine nodular dermatosis.In this study,the target gene ORF132 was optimized by codon and recombinant baculovirus(ACMP NPV-ORF 132 and ACMP NPV-Y-ORF 132)was constructed by screening the target gene.AcMNPV-ORF132 and AcMNPV-Y-ORF132 can be expressed in Sf9 cells.The protein size detected by His tag antibody is about 26 KD,which is 6KD larger than expected.The codon-optimized AcMNPV-Y-ORF132 has higher expression than the non-optimized AcMNPV-ORF132.After codon optimization,the Codon Adaptation Index,CAI)of Y-ORF132 increased from 0.62 to 0.98,the G+C content increased from 25% to 52%,and the most biased codon ratio increased from 35% to 89%,which is more suitable for insect baculovirus expression.By measuring the proliferation curve and virus titer of AcMNPV-Y-ORF132,its virus quantity and titer increase with time.The expression of ORF132 recombinant protein in Sf9 cells was proved by indirect immunofluorescence.In order to explore the expression conditions of AcMNPV-Y-ORF132 in Sf9 cells,the number of inoculated cells,virus multiplicity of infection and virus infection time were optimized.Through Western-Blot analysis,the optimal cell inoculation density was 1.6×106 cells/ml.The best multiplicity of infection is 1;The best time to harvest protein was 96 hours.In this experiment,AcMNPV-Y-ORF132 infected silkworm was tested for nucleic acid in silkworm body by qPCR.The results showed that Y-ORF132 recombinant baculovirus could replicate and proliferate in silkworm body.The purified AcMNPV-Y-ORF132 particles were injected intraperitoneally to immunize mice.The results showed that the Y-ORF132 recombinant baculovirus particles had good immunogenicity through the determination of spleen lymphocyte,specific antibody and antibody titer.