Development And Preliminary Application Of Monoclonal Antibody Against Lawsonia Intracellularis Hsp60 Protein

Lawsonia Intracellular(LI)caused porcine proliferative enteritis(PPE),which was first reported in Britain in 1931,followed by repeated reports around the world in the following decades.Acute hemorrhagic hypertrophic enteritis usually occurs in young pigs between 4 and 12 weeks and presents as hemorrhagic enteritis with sudden death.Chronic infection often occurs in piglets between 6 and 20 weeks.In addition to slow growth and development,there are no obvious clinical symptoms.The pathogen was difficult to isolate,and the successful isolation was only reported in 1993.Until now,the research on the pathogenic mechanism of LI is very limited,which seriously affects the prevention and treatment of porcine proliferative enteritis.In this study,monoclonal antibody was prepared by expressing Hsp60 protein of this bacterium,and an IFA detection method was established,providing an effective means for rapid diagnosis of this disease.1.Development and identification of recombinant proteinsBased on the antigenicity of bioinformatics analysis,this study choose Hsp60 gene(GenBank:BAE54309.1)as a purpose,using PCR amplification its total length of 1647 bp,pET28a-Hsp60 recombinant plasmid was constructed,after waiting for sequencing right it into E.coli BL21 cells,using 1 mm concentration of IPTG as inducers,37℃ 8 h,induced successfully expressed its recombinant protein in vitro,and the recombinant protein solubility is good,through SDS-PAGE analysis,the recombinant protein molecular weight was 60.3 KD,with expectations.The antigenicity of the recombinant protein was analyzed by Western-blot analysis.2.Development of monoclonal antibodyThe purified recombinant protein was used as antigen to immunize Balb/c mice at the age of 10 weeks,and the dose was 50 μg/mouse.Through a series of routine monoclonal antibody preparation procedures,three hybrid tumor cell lines,named 4F,9G and 6E,which could secrete specific antibodies steadily were obtained.The three hybrid tumor cell lines were used to immunize mice,and antibodies against ascites were successfully prepared and collected.After purification of antibodies against ascites,indirect ELISA was used to determine the titer of antibodies against ascites,4F was 1:213,9 G was 1:213,6 E was 1:211.3.Establishment and application of IF A methodThe IFA method for LI was successfully established by using hybrid tumor cell line 9G.This method has strong specificity and no cross reaction with other intestinal viruses PEDV and TGEV in pigs,with good repeatability and sensitivity of 102 cell/mL,better than the fecal PCR.Twenty-five clinical samples were tested by IFA,the positive rate was 28%,and the coincidence rate with PCR was 100%.