Cloning and expression of human filaggrin gene and detection of anti-filaggrin antibodies for diagnosis of rheumatoid arthritis

Abstract

Objective To construct the recombinant plasmid containing human filaggrin gene,purify and identify the immunoreactivity of the recombinant protein,and establish the indirect ELISA to detect AFA for diagnosis of RA.Methods The constructed plasmids were transformed into E. Coli Rosettagami(DE3).This fusion protein was purified by NAT chromatography.ELISA coated with the fusion protein Was established to detect the AFA in serum of patients,which included 114 cases of RA,56 cases of SLE,32 cases of OA and 40 cases of normal controls. The correlation between the results of AFA and anti-CCP in RA group were compared. Results 321 bp fragment of filaggrin gene was amplified and the recombinant expression vector pET-28a( + )-filaggrin was constructed. The sequence of filaggrin gene was the same as the sequence reported in the literatures. The Rosetta-gami (DE3) strains of E. Coli with recombinant vector showed high level of filaggrin protein after induction. The SDS-PAGE showed that the plasmid expressed the filaggrin fusion protein with molecule weight of 14 000 Da. The expression protein could be purified by Ni-NAT with activity. The absorbance value of AFA in RA group was 0.473 ±0. 248 while they were 0. 160 0. 088, 0. 121±0. 070, 0.050 0. ±018 in SLE, OA and normal groups respectively. There were significant differences of absorbance values of AFA between RA and SLE, OA, control group (t = 12.004, 14. 464, 18.078, P<0. 01, respectively). The positivities of anti-filaggrin in RA, SLE and OA were 48.2%, 5.4% and 3. 1% respectively. The positivities of AFA were significantly different between RA, OA and normal control groups (x~2 = 67. 088, P < 0. 01). There was positive correlation of results between AFA and anti-CCP antibody (r = 0.42, P < 0. 05 ) . The consistency rate of results between AFA and anti-CCP was 70. 1%. Anti-CCP was negative in 10 out of 114 patients with AFA positive. AFA can be used to diagnose RA with sensitivity of 48. 2% , specificity of 96.9% , positive predictive value of 93. 2% and negative predictive values of 67. 9% . Conclusions The purified human filaggrin fusion protein is successfully purified. The indirect ELISA method based on the recombinant protein shows good sensitivity and specificity. Joint detection with AFA and anti-CCP can improve the positive rate of detection. Key words: Arthritis, rheumatoid; Intermediate filament proteins; Enzyme-linked immunosorbent assay