CAJ | 학술논문

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321 bp人filagrin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Rosettagami(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14 000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、0A组及健康对照组的A值(分别为0.160±0.088、0.050±O.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(x~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fitaggrin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.
목적 통과극륭인취각미사단백(filaggrin)기인,구건중조표체질립,획득구활성적순화중조단백,건립인AFA간접ELISA검측법,이평개AFA재RA진단중적개치.방법 구건중조표체재체,재대장간균Rosetta-gami(DE3)중표체;융합단백경Ni-NAT수지주진행친화층석순화,건립간접ELISA검측AFA방법,병용우검측114례RA환자、56례SLE환자、32례OA환자급40명건강체검자혈청중항인AFA;분석AFA화항CCP항체진단RA적상관성.결과 확증출321 bp인filagrin기인편단,구건료구유정학서렬적질립재체pET-28a(+)-filaggrin,전화대장간균Rosettagami(DE3)중경유도산생고수평적표체산물.경SDS-PAGE분석,재상대분자질량위14 000처출현신생단백대.용Ni-NAT수지순화후득도구유활성적순화인filaggrin중조단백.간접ELISA검측표본혈청결과현시,RA조AFA검측흡광도(A)치위0.473±0.248,여SLE조、0A조급건강대조조적A치(분별위0.160±0.088、0.050±O.018、0.121±0.040)량량조간비교,차이구유통계학의의(t치분별위12.004、14.464、18.078,P균<0.01).AFA재RA조、SLE조급OA조양성솔분별위48.2%、5.4%화3.1%.RA조AFA양성솔여SLE조、OA조급건강대조조비교,차이유통계학의의(x~2=67.088,P<0.01).AFA여항CCP항체대RA적진단정정상관(r=0.42,P<0.05).AFA여항CCP양성일치솔위70.1%,114례환자중유10례환자항체CCP음성,이AFA검측결과위양성.AFA대RA진단적민감도、특이도、양성예측치화음성예측치분별위48.2%、96.9%、93.2%화67.9%.결론 용순화fitaggrin융합단백건립적간접ELISA검측항인AFA적방법,대RA진단구유교호민감도화특이도,AFA여항CCP항체공동검측가제고검측양성솔.
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.