Cloning, Expression And Antigenicity Analysis Of Mycobacterium Bovis Major Secreted Immunogenic Protein 70 And Lipoprotein 83

Since 1882, when German bacteriologist Koch firstly discovered Mycobacteriumtuberculosis, more than 100 Mycobacterium species have been reported.Mycobacteria are generally classified into two classes. One is Tuberculosismycobacterium (TM), also called Mycobacterium tuberculosis complex (MTBC),including M. tuberculosis, M. bovis and M. africanum. The other is Non-tuberculosismycobacterium (NTM), also called atypical mycobacteria, including all mycobacteriaexcept MTBC and M. leprae.M. bovis is widely transmitted among both humans and animals. Recently, risingM. bovis infections in human have become a severe public health problem. M. bovishas many animal hosts, such as cattle, badgers and deer. Infected animals can pollutethe environment by sputum, milk, feces. Fast and accurate diagnosis is important forcontrol of bovine tuberculosis. Purified protein derivative (PPD) sensitive skin test(TST) is currently being used for diagnosis of bovine tuberculosis. It takes about 48hours to obtain results. In M. bovis endemic regions, some healthy cattle are oftenmisguidely euthanized due to false-positive results of TST, because of a nonspecificinflammatory response to PPD and cross-reactions with nonpathogenic species ofenvironmental mycobacteria.In recent years, colloidal-gold immunochromatography assay (GCIA) hasattracted close attention due to its quickness and convenience. Colloidal-gold is goldparticles of specific sizes, produced by reducing reagents, such as Vitamin C, ascorbicacid sodium, sodium citrate. Under certain conditions, negatively chargedcolloidal-gold particles can combine Staphylococcal protein A (SPA), IgG and manyother proteins. This combination has no adverse effects on proteins'biologicalactivity.Major secreted immunogenic protein 70 (MPB70) and cell surface lipoprotein 83(MPB83) are major antigens of M. bovis. In early stages of M. bovis infection, bothMPB70 and MPB83 can induce both cellular and humoral immune responses.Anti-MPB70 and Anti-MPB83 antibody levels in serum are positively correlated withdisease progression. These two major antigens are highly expressed in M. bovis, butconsiderably less abundantly expressed in M. tuberculosis. Detection of MPB70 orMPB83 antibodies is useful for diagnoses of M. bovis infections.In this study, mpb70 and mpb83 genes of M. bovis were amplified by polymersechain reactions (PCR), inserted into pET-32a(+) vector (pET-32a(+)-mpb70 andpET-32a(+)-mpb83) and transformed into E. coli BL21 (DE3). The target genes wereconfirmed to be in correct reading frame in plasmids pET-32a(+)-mpb70 and pET-32a (+)-mpb83 by DNA sequencing. Sequencing results revealed that the nucleotidesequences of both cloned genes are 100% identity with those of M. bovis AF2122/97reference strains. After IPTG induction, recombinant MPB70 and MPB83 weremainly expressed in inclusion bodies with a high yield of about 60%. Combined withpET-32a (+) expression vector-fused thioredoxin and S protein, the molecular weightsof rMPB70 and rMPB83 are predicted to be 42kDa, 45kDa respectively. SDS-PAGEanalyses indicated that the actual molecuar weights of rMPB70 and rMPB83 arecorrect. After purification, the purity of both rMPB70 and rMPB83 was over 80%.Western blot assays demonstrated that rMPB70 and rMPB83 proteins couldspecifically react with serum of bovine tuberculosis, indicating good antigenicity ofboth rMPB70 and rMPB83.Primary applications of purified rMPB70 and rMPB83 in GCIA were furtherexplored. Colloidal-gold was prepared by reduction of a gold salt with sodium citrate.SPA can combine a variety source of mammalian IgG. SPA was labeled withcolloidal gold. Nitrocellulose (NC) membrane was sensitized with variousconcentrations of rMPB70 and rMPB83, respectively. The optimal concentrations ofrMPB70 and rMPB83 for NC membrane sensitization were 4.5mg/ml and 3mg/ml,respectively. In our preliminary experiment, normal bovine sera and normal salineshowed negative results, while the result with TST-positive bovine sera was different.,20 (66%) showed positive results with rMPB70 in 30 TST-positive cattle sera samples,while 22 (73%) showed positive results with rMPB83. Our data suggested that bothrMPB70 and rMPB83 are useful antigens for serological diagnosis of bovinetuberculosis.