Preparation Of Monoclonal Antibodies To The LI0902 Protein Of Lawsonia Intracellularis And Establishment And Preliminary Application Of Indirect ELISA Method

Porcine proliferative enteritis(PPE)is caused by Lawsonia Intracellularis(LI)infection and then causes the "flexible bowel" disease that causes the characteristic hyperplasia mainly in the ileum.Porcine proliferative enteritis makes it difficult for a large number of immature pig intestinal crypt cells to ensure normal nutrient absorption,resulting in a decrease in feed conversion rate and weight loss,which seriously threatens the healthy development of the pig industry.The whole genome size of the bacteria About 1.4Mb,there are five outer membrane proteins expressed during infection,of which LI0902 is one of them,and it is highly expressed in the cytoplasm of diseased tissues,and may be involved in adhesion,invasion and colonization in the process of pathogenicity.In order to better prevent and control the disease and lay a foundation for further research on the disease,the main contents include:1.Construct Gst tag plasmid and purify the target protein containing His and Gst tag LI0902Lawsonia intracellularis reference strain PHE/MN1-00 sequence,design LI0902 gene(accession number: AM180252.1),the recombinant strain p GEX-4T-1-LI0902/BL21(DE3)and Culture based on the constructed recombinant strain p ET32a-LI0902/BL21(DE3),carry out mass expression and purification,and obtain His-LI0902 protein and LI0902-Gst protein.2.Preparation of monoclonal antibody based on His tag LI0902 protein of Lawsonia intracellularisBased on the recombinant Lawsonia His-LI0902 protein as the antigen,BALB/c mice were immunized.After evaluating the effectiveness of the antibody by the ELISA method,the mice with higher titer were used for cell fusion,and the His-LI0902 and Gst-LI090 proteins were not antigens.The Western Blot method successfully screened 4 strains against Lawsonia intracellularis LI0902 The protein cell lines were named 1G1-2,2G8,4A4,2H4,respectively.Up to now,there are no monoclonal antibodies that have been applied to clinics.In this study,four monoclonal antibody cell lines were successfully prepared,which lays the foundation for further research on Lawsonia intracellularis in the future.3.Establishment of indirect ELISA method3.Establishment of indirect ELISA methodThe antigen is the recombinant Lawsonia intracellularis His-LI0902 protein,and the enzyme-labeled secondary antibody is goat anti-pig HRP-Ig G.The optimal conditions are explored and the indirect ELISA method is successfully established.After preliminary evaluation of various conditions: the coated antigen concentration is 2.5μg/m L;in addition,it is found that the appropriate dilution ratio of serum dilution is 1:32000 times and the dilution ratio of goat anti-pig HRP-Ig G is 1:2000 times,and the blocking time Both are 60minutes;the blocking solution is 1% BSA for 120 minutes;the threshold of yin and yang serum is 0.334;the intra-assay and inter-assay coefficients of variation are 1.1335%-8.364%and 1.167%-4.694%,respectively;Random samples were taken from the field for serological testing of Lawsonia intracellularis,and the average positive rate was 25.3%.The indirect ELISA method established in this experiment has good repeatability and specificity,and provides technical support and auxiliary tools for future epidemiological investigations,clinical testing,diagnosis and vaccine immunity.clinical testing,diagnosis and vaccine immunity.