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The gene coding for an antigenic 34-kDa protein of Mycobacterium paratuberculosis was isolated and sequenced. The 897-bp open reading frame coded for a novel protein containing specific B epitopes. The occurrence of well-defined hydrophobic and hydrophilic portions suggests the wall location of the protein.Mycobacterium paratuberculosis, the etiological agent of Johne's disease, contains a major antigen complex called A36 which is highly recognized by sera from cattle infected with M. paratuberculosis (6,9). We (18), and the insert (boxed in Fig. 1) was sequenced with the universal primers SK and KS (Stratagene) and the T7 sequencing kit used with Deaza G/A T7 sequencing mix (Pharmacia, Uppsala, Sweden).The gene sequence revealed two open reading frames, possibly in phase with the ,-galactosidase gene of Xgtll, at each extremity of the inserted mycobacterial DNA. In one direction, a stretch of 299 bp (nucleotides 1340 to 1638 in Fig. 1) corresponding to a 13.6-kDa polypeptide was found, whereas in the opposite direction there was a 179-bp frame (nucleotides 1832 to 1654 in Fig. 1) coding for a 7.2-kDa polypeptide. The original orientation of the inserted mycobacterial DNA fragment in phage Xgtll-a362 (unidentifiable after its transfer into the sequencing vector) was established as follows. The mycobacterial DNA insert was cloned in both orientations into the expression plasmid pmTNF.MPH, and both reading frames were translated (Fig. 2). This vector allows the expression of cloned genes as fusion proteins with a polypeptide comprising the first 25 amino acids of mouse tumor necrosis factor and a hexahistidine peptide (4). The resulting recombinant plasmids were introduced into E. coli K-12 A H1 (ATCC 33767) by transformation (18). Single * Corresponding author. transformed colonies were grown at 28°C in the presence of tetracycline to an A6. of 0.2 and then heat shocked (4 h, 42°C). Cell lysates were tested, in Western blot (immunoblot) experiments, with rabbit antiserum directed against the fusion protein expressed by phage Xgtll-a362. Restriction analysis of positive clones (pmTNF.MPHa362) indicated that the correct orientation of the mycobacterial DNA fragment in phage Xgtll-a362 involved the transcription of the 299-bp open reading frame, which encoded a 13.6-kDa polypeptide.Confirmation of this result, and of the corresponding nucleotide sequence, was obtained by sequencing the purified recombinant polypeptide. A sonic extract of the induced recombinant bacteria containing pmTNF.MPHa362 was centrifuged (25,000 x g, 15 min, 4°C), and supernatant proteins were precipitated [30% (wt/vol) (NH4)2SO4], dissolved (0.5 M NaCl-15 mM potassium phosphate buffer, pH 5.2), and dialyzed overnight at 4°C (against the same buffer). Guanidine chloride was added (final concentration, 4 M in 50 mM phosphate buffer, pH 7.4), and the recombinant polypeptide carrying six adjacent histidines (Fig. 2) was purified from E. coli proteins by selective interaction between the polyhistidine peptide and a metal chelate adsorbent (Ni...

31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease); an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70o%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40%o of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle.

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