人聚角微丝蛋白基因克隆表达及其抗体在类风湿关节炎诊断中的价值

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

目的通过克隆人聚角微丝蛋白（filaggrin）基因，构建重组表达质粒，获得具活性的纯化重组蛋白，建立人AFA间接ELISA检测法，以评价AFA在RA诊断中的价值。方法构建重组表达载体，在大肠杆菌Rosetta—gami（DE3）中表达；融合蛋白经Ni—NAT树脂柱进行亲和层析纯化，建立间接ELISA检测AFA方法，并用于检测114例RA患者、56例SLE患者、32例OA患者及40名健康体检者血清中抗人AFA；分析AFA和抗CCP抗体诊断RA的相关性。结果扩增出321bp人filaggrin基因片段，构建了具有正确序列的质粒载体pET-28a（＋）-filaggrin，转化大肠杆菌Rosetta—gami（DE3）中经诱导产生高水平的表达产物。经SDS—PAGE分析，在相对分子质量为14000处出现新生蛋白带。用Ni—NAT树脂纯化后得到具有活性的纯化人filaggrin重组蛋白。间接ELISA检测标本血清结果显示，RA组AFA检测吸光度（A）值为0．473±0．248，与SLE组、OA组及健康对照组的A值（分别为0．160±0．088、0．050±0．018、0．121±0．040）两两组间比较，差异具有统计学意义（t值分别为12．004、14．464、18．078，P均〈0．01）。AFA在RA组、SLE组及OA组阳性率分别为48．2％、5．4％和3．1％。RA组AFA阳性率与SLE组、OA组及健康对照组比较，差异有统计学意义（χ^2=67．088，P〈0．01）。AFA与抗CCP抗体对RA的诊断呈正相关（r=0．42，P〈0．05）。AFA与抗CCP阳性一致率为70．1％，114例患者中有10例患者抗体CCP阴性，而AFA检测结果为阳性。AFA对RA诊断的敏感度、特异度、阳性预测值和阴性预测值分别为48．2％、96．9％、93．2％和67．9％。结论用纯化filaggrin融合蛋白建立的间接ELISA检测抗人AFA的方法，对RA诊断具有较好敏感度和特异度，AFA与抗CCP抗体共同检测可提高检测阳性率。

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. eoli 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 （χ^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.