Immune Cross-reaction Of Ag85b Proteins Between Ycobacterium Bovis And Nontuberculous Mycobacteria

Bovine tuberculosis (BTB) is an infectious chronic respiratory caused by Mycobacterium bovis(M.bovis). It seriously inhibit the development of animal husbandry and threaten to human health. Inaddition, there are many Nontuberculous Mycobacterium (NTM) which caused symptoms like that oftuberculosis, which posed difficulties to the diagnosis and treatment of M.bovis. Ag85b is the secretedproteins of Mycobacteria, which is an important protective antigen of BTB, and it can enhance theTh1-type cellular immune response characterized by interferon-gamma (IFN-γ) production. Ag85bprotein mainly existes in Mycobacterium tuberculosis (MB) or M.bovis, and it also exists in the otherNTM. So the study on the immune cross-reaction of Ag85b proteins between M.bovis and NTM is ofgreat significance.In this study, Ag85b genes were amplified by PCR from genomes of Mycobacterium aviumsubsp.paratuberculosis(M.paratuberculosis), Mycobacterium smegmatis (M.smegmatis),Mycobacteriummarinum (M.marinum), Mycobacterium bovis (M.bovis), Mycobacterium avium (M.avium)serotype2,respectively, subcloned into pET28a and pcDNA3.1. Induced expression of the five kinds ofrecombinant Ag85b(rAg85b) proteins in E.coli and then purify the rAg85b proteins by Ni-NTA His bindresin. Guinea pigs were immunized with Eukaryotic expression recombinant plasmids of five Ag85bgenes (pcA) repectively, five rAg85b proteins are as coating antigens to analyse the cross-reactionamong five prepared anti-Ag85bs. Take the spleen lymphocytes of guinea pigs immunized with pcA,and stimulate the lymphocytes with five rAg85b proteins, then tested the splenic lymphocytesproliferation (SLP) and secretion of IFN-γ.In the study we have obtained five rAg85b proteins detected by Western blot and the results ofindirect ELISA showed that there are strong serological cross-reaction of Ag85b proteins among fivemycobacteria, SLP experiments indicate that each kind of rAg85b protein can stimulate the proliferationof lymphocytes of every experimental group and the IFN～γ experiments showed that the levels ofIFN～γ induced with rAg85b in each group are significantly higher than control group (p <0.01).These studies show that there are serious cross-reaction of Ag85b proteins amongM.paratuberculosis, M.smegmatis, M.marinum, M.bovis, and M.avium serotype2, which will pose greatimpact on the immune monitoring of Ag85b as a candidate vaccine. So it should be add an identifiedmarkers to identify vaccination and natural infection when Ag85b is as a new BTB vaccine, which willbe important for vaccine monitoring and purification of cattle. At the same time, the study also indicatedthat Ag85b protein may provide cross-protection on M.bovis and NTM infection, and it can be also usedas a good anti-tuberculosis vaccine adjunct to enhance the immune response against M.bovis and NTMinfection.