Even though pathophysiological role of the N46 peptide remains to be determined, this new antigenic target appears to be implicated in pSS. anti-N46 peptide antibodies (-N46PA) in patients with pSS, SLE, RA and normal controls were 78.5%, LATS1/2 (phospho-Thr1079/1041) antibody 10.4%, 21.6% and 6.0%, respectively. The sensitivity and specificity of the autoantibodies in pSS were 78.5% and 86.8%, respectively. These results suggest the -N46PA which shows highest sensitivity and specificity is usually of significance to develop an effective diagnostic approach for Zileuton pSS. BL21 and purified by affinity chromatography, thrombin digestion and gel filtration by Sephacryl S100 column. As shown by SDS-PAGE, glutathione S-transferase (GST)-fused -fodrin N-terminal fragments amino acids 1C59, 1C128, 1C167, 1C238, 1C284, 1C360, 1C423 and 1C485 Zileuton were prepared with high purity ( 95%) (Fig. 2b). Open in a separate windows Fig. 2 The fragments of a series of -fodrin cDNA (a) constructs amplified by polymerase chain reaction (PCR). M. 100 base pairs (bp) DNA ladder marker. 1C8. The eight overlapped cDNA segments of -fodrin. These fragments were prepared from your 1785 cDNA including 1680 cDNA of -fodrin using specific primer pairs, including 1C1560 (1), 1C1374 (2), 1C1185 (3), 1C957 (4), 1C819 (5), 1C606 (6), 1C489 (7) and 1C282 bp (8). Sodium dodecyl sulphateCpolyacrylamide gel electrophoresis (SDS-PAGE) (b) analysis of expression and purification of the eight overlapping protein segments of -fodrin 560. 1C8. The purified recombinant -fodrin overlapping proteins, which encompassed aa 1C485 (1), 1C423 (2), 1C360 (3), 1C284 (4), 1C238 (5), 1C167 (6), 1C128 (7) and 1C59 (8), respectively, of -fodrin Zileuton 560. Immunoassays (c) for the recombinant eight recombined overlapping fusion proteins of -fodrin 560 by Western blot. 1C8. The result of Western blot of eight recombined overlapping fusion proteins of -fodrin 560 on a mixture of five anti–fodrin antibody-positive Zileuton pSS sera. G. The result of Western blot of glutathione S-transferase (GST) protein on the same mixture of five anti–fodrin antibody-positive pSS sera. N. The result of Western blot of the recombined overlapped fusion protein segments combination on a mixture of five anti–fodrin antibody-negative main Zileuton Sj?gren’s syndrome (pSS) sera. Immunoresponses of the recombinant overlapping proteins to pSS sera and the determination of the epitope region Figure 2c showed that all recombinant overlapping proteins had strong immunoresponses to the mixture of five anti–fodrin antibody-positive pSS sera. The major immunoreactive bands appeared close to the respective molecular weight of the overlapping proteins of the theoretical estimation. Because comparable immunoreactivities were observed in all the eight overlapping protein recombinants, they might share a similar epitope site, possibly within the N-terminal 59 amino acids (N59) of -fodrin 560, which are included in all other recombinant fragments. Moreover, the protein segment of -fodrin, except the possibly shared epitope amino acids (1C59 aa), was expressed and detected with Western blot above (Fig. 3aCc). The results showed that this recombinant protein segment experienced no immunoresponses to the same serum combination samples as above; the antigenic determinant region of -fodrin 560 was then acknowledged from codons 1C59 aa. Open in a separate windows Fig. 3 Polymerase chain reaction (PCR) amplification (a) of the gene of -fodrin except the possibly shared epitope gene [1C282 base pairs (bp)]. 1. 100 bp DNA ladder marker. 2. The PCR DNA of part of the -fodrin except the possibly shared epitope gene (1C282 bp). Sodium dodecyl sulphateCpolyacrylamide gel electrophoresis (SDS-PAGE) analysis (b) of expression and purification of the protein segment of -fodrin except the possibly shared epitope amino acids (1C59.