Application Of DNA Immunization In The Preparation Of Secondary Antibodies And Immunogen Evaluation Of Tree Shrews

Objective:Currently,tree shrews have become a potential animal model for studying human diseases,but there is a lack of corresponding humoral immunoassays,and the expression of tree shrew IgE recombinant antibodies cannot be purified after transfection expression.Therefore.the DNA immunization method can be chosen to prepare the tree shrew IgE secondary antibody simply and quickly,and the step of purifying the antibody can be omitted,and the tree shrew secondary antibody can be prepared by DNA immunization directly.The gene gun technique in DNA immunization has the characteristics of fast and efficient.The DNA immunoassay technique is fast and efficient,and it can facilitate the evaluation of the immunogenicity of different lengths of DNA expressing neocrown thorny proteins in experimental animals.Method:The gene of the antibody was recombinant using recombinant DNA technology;the target gene was cloned into the expression vector and transfected into HEK293T cells to express recombinant tree shrew IgE antibody,and the protein expression was examined by WB,and the immunization was performed by DNA immunization method assisted by gene gun(using BIORAD Helios Gene Gun)for female weighing 2-2.5kg The amount of DNA was 1 μg/bullet,10 bul lets for each of th e first and second immunization,20 bullets for the third immunization,and serum was collected from the immunized animals after the three immunizations.The tree shrew secondary antibody was prepared simply and quickly.In the second part,the Helios Gene Gun was used for immunization with different lengths of plasmids expressing novel coronavirus spike-in proteins in the amount of 5 μg DN A per bullet.Each tree shrew was immunized with 1 shot per Gene Gun,and the immunization site was the hind leg of the tree shrew,with 1 shot per leg.Blood was collected before each immunization and 2 weeks after the third immunization for the detection of IgG and neutralizing antibodies in the serum.Results:A plasmid containing the tree shrew IgE gene was successfully constructed,and the plasmid was transfected into HEK293T cells to suc.cessfully express the tree shrew recombinant IgE antibody,but the expression amount was not high and the tree shrew recombinant IgE antibody could not be purified.Therefore,a gene gun-assisted DNA immunization method was used to immunize female Japanese large-eared white rabbits weighing 2-2.5 kg directly with a gene gun bullet containing gold particles,and yellow-brown spots were formed at the gene gun attack after immunizing the rabbits.After the third immunization,the rabbit serum was diluted]:500 with PBST and the tree shrew recombinant IgE antibody was detected,and there was no cross-reaction with the tree shrew recombinant IgM and IgA antibodies.Also in the second part,IgG antibodies to anti-crown S1 protein were detected in sera after immunization of tree shrews with different lengths of plasmids expressing novel coronavirus spike-in proteins using a gene gun.No antibody response was evident after the first immunization,nearly half of the tree shrews produced antibody responses after the second immunization,and after the third immunization,all tree shrews in the full-length S protein group were positive for anti-new coronavirus S1 IgG antibodies.The results of neutralizing antibodies showed that no neutralizing antibodies were evident after the first immunization,and neutralizing antibodies started to be produced in the tree shrews after the second immunization,and after the third immunization,all tree shrews produced anti-SARS-CoV-2 neutralizing antibodies.Conclusion:The gene gun-assisted DNA immunization method allows for the acquisition of tree shrew secondary antibodies after the third immunization.By using DNA immunization,the immunogenicity of different novel coronavirus spike-in protein sequences can be rapidly evaluated,thus providing immune reference data for the development of relevant new coronavirus vaccines.