Construction Of Prokaryotic Expression System Of IBDV Single Chain Antibody And Analysis Of Its Antiviral Effect

Infectious Bursal Disease（IBD）is a highly contagious and highly infectious disease that mainly infects chickens with Infectious Bursal Disease Virus（IBDV）as the pathogen.The disease has an acute course of disease and a rapid spread.IBDV mainly caused a large number of necrotic reduction and apoptosis of B lymphocytes,resulting in immunosuppression and death of chickens.IBD has caused huge economic losses to the global poultry industry since its outbreak.In recent years,gene mutation and antigenic drift have led to the generation of mutant strains and super-virulent strains,which has brought new challenges to the prevention and treatment of infectious bursal disease.Single chain antibody is a genetically engineered antibody with a small molecular weight,which has the ability to specifically bind to an antigen because its antigen-binding site is the same as that of an intact antibody.The molecular weight of single-chain antibody is only about 25 KDa,so it can pass through the blood vessel wall and tissue barrier.Since single chain antibodies do not contain redundant structures of complete antibodies,they have low immunogenicity,and can be modified by molecular biology methods to prepare genetically engineered antibodies.Based on these characteristics,in this study,a single chain antibody CRAb20with the ability to neutralize IBDV,and its gene sequence was obtained.E.coli and Lactobacillus expressing CRAb20 were constructed and their protective effects against IBDV infection in chickens were detected.The main results are as follows:In this experiment,by constructing E.coli expressing CRAb20,the target single chain antibody pCold-CRAb20 with a molecular weight of about 100 KDa was obtained by soluble expression,and purified by His affinity chromatography column to remove impurity proteins.The purified pCold-CRAb20 was used to immunize depleted BALB/c mice to prepare polyclonal antibodies,and the mouse anti-CRAb20 polyclonal antibody with a titer of 1:12800 was obtained for validation experiments.A chicken-derived Lactobacillus expressing CRAb20 was constructed,and CRAb20 was expressed and secreted into the supernatant of recombinant Lactobacillus culture.The target single chain antibody pPG-CRAb20 with a size of about 43 KDa was obtained.The binding ability and neutralization of CRAb20 expressed by recombinant bacteria and vvIBDV LJ-5 were tested in vitro.It can be seen that CRAb20 expressed in both forms can specifically bind to vvIBDV LJ-5 and inhibit the proliferation of vvIBDV in DT40 cells.In order to detect the anti vvIBDV LJ-5 infection effect of CRAb20 on chickens,SPF chickens were selected and divided into prevention group,treatment group,challenge control group and PBS control group.7 days after challenge was set as the observation period.The prevention group was divided into pCold-CRAb20 prevention group(Chickens at age of 4 weeks were challenged with 10³ELD₅₀LJ-5,intranasally and intraocularly with 100μL,orally with 100μL.Each chicken was injected 5 mg purified pCold-CRAb20 24 h before challenge),pPG-CRAb20prevention group and pPG empty carrier bacteria group(Chickens were fed with 5×10⁹CFU/mL Lactobacillus at age of 19 days for 2 consecutive days with 1 day interval and repeated three times.Chickens at age of 4 weeks were challenged with 10³ELD₅₀LJ-5,intranasally and intraocularly with 100μL,orally with 100μL.).The treatment group was divided into pCold-CRAb20 treatment group(Chickens at age of 4 weeks were challenged with 10³ELD₅₀LJ-5,intranasally and intraocularly with 100μL,orally with 100μL.Each chicken was injected 5 mg purified pCold-CRAb20 24 h after challenge until the end of the observation period),pPG-CRAb20 treatment group(Chickens at age of 4 weeks were challenged with 10³ELD₅₀LJ-5,intranasally and intraocularly with 100μL,orally with 100μL.Chickens were fed with 5×10⁹CFU/mL Lactobacillus until the end of the observation period).Chickens were divided into challenge control group(Chickens at age of 4 weeks were challenged with 10³ELD₅₀LJ-5,intranasally and intraocularly with 100μL,orally with 100μL.),PBS control group（19-day-old chickens were selected and fed with sterile PBS during the experiment）.During the observation period,the chickens are monitored for weight changes and mortality.The dead chickens were necropsied,and the bursa was taken for fixation to prepare pathological sections,and the lesion degree of the bursa was observed.The total RNA in bursa,spleen and thymus was extracted,and the viral load in tissues was detected by RT-qPCR.Venous blood was collected from chickens with obvious symptoms,and serum was collected to detect the expression levels of IL-6,TNF-α,IFN-γ,IL-10 and TLR3.The results showed that compared with PBS control group,the weight of chickens in pPG-CRAb20 prevention group and pPG empty carrier bacteria group increased significantly,showing the growth promoting effect of lactic acid bacteria on chickens.After the challenge,the body weight of the chickens in the challenge control group and the pPG empty carrier bacteria group decreased sharply,while the body weight of the prevention group and the treatment group decreased slowly.Comparing the average bursa weight,bursa weight ratio,and BBIX of chickens in each group,it was found that compared with the PBS control group,the average bursa weight and bursa weight ratio in the challenge control group and the pPG empty carrier bacteria group decreased significantly（P<0.01）,BBIX<0.7.The average weight of the bursa and the bursa weight ratio of the prevention group and the treatment group decreased slightly,and the BBIX was basically greater than 0.7.Histopathological observation showed that a large number of lymphocyte necrosis and lymphoid follicle atrophy occurred in the bursal tissue of the challenge control group and the pPG empty carrier bacteria group,accompanied by hyperplasia of connective tissue,infiltration of heterophilic cells,and structural collapse of the bursa of Fabricius.The pathological changes in the bursa of the prevention group were not obvious.There was a small amount of lymphopenia and lymphoid follicle atrophy in the bursa of the treatment group.PBS control group showed no pathological changes.The viral loads in the bursa,spleen,and thymus were detected.Compared with the challenge control group and the pPG empty carrier bacteria group,the viral loads in the tissues were significantly decreased（P<0.01）.Comparing the viral loads of the three groups,it was found that the bursa was the main organ infested by vvIBDV.The expression levels of cytokine in chick serum were detected by ELISA.Compared with those in the challenge control group and the pPG empty carrier bacteria group,the results showed that the expression levels of IL-6 and TNF-αwere significantly decreased（P<0.01）,and the expression levels of IFN-γ,IL-10,and TLR3 were significantly increased（P<0.01）.During the observation period of 7 days after the challenge,the chickens in the recombinant bacteria prevention group and the treatment group began to die on the 5th day after infection,and the chickens in the challenge control group and the pPG empty carrier bacteria group began to die on the 4th day after infection.The survival rate of pPG-CRAb20 prevention group was 28.57%,the survival rate of pCold-CRAb20 prevention group was 42.86%,the survival rate of pPG-CRAb20 treatment group was 14.29%,and the survival rate of pCold-CRAb20 treatment group was 28.57%.All the chickens in the challenge control group and the pPG empty carrier group died,and all the chickens in the PBS control group survived.In conclusion,CRAb20 expressed by recombinant E.coli and recombinant chicken-derived Lactobacillus can specifically bind to vvIBDV LJ-5 strain and inhibit the proliferation of vvIBDV LJ-5 strain on DT40 cells.Feeding recombinant chicken-derived Lactobacillus expressing pPG-CRAb20 and injecting pCold-CRAb20 expressed by recombinant E.coli can reduce the damage of vvIBDV to chickens’bursa,improve the survival rate,and have certain preventive and therapeutic effects on chickens’anti-IBDV infection.