中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2013年
5期
305-308
,共4页
谢小平%刘双全%张秋桂%吴移谋
謝小平%劉雙全%張鞦桂%吳移謀
사소평%류쌍전%장추계%오이모
密螺旋体,苍白%重组蛋白质类%TP0993%免疫活性
密螺鏇體,蒼白%重組蛋白質類%TP0993%免疫活性
밀라선체,창백%중조단백질류%TP0993%면역활성
Treponema pallidum%Recombinant proteins%TP0993%Immunocompetence
目的 探讨梅毒螺旋体重组蛋白TP0993在梅毒血清学诊断中的应用价值.方法 通过生物信息学分析,获取TP0993基因序列,构建原核载体进行诱导表达;Ni-NTA亲合层析柱纯化重组蛋白,Western印迹检测其免疫抗原性,用重组蛋白直接注射免疫新西兰家兔,评价其免疫原性.用纯化的TP0993重组蛋白包被微孔板,建立间接酶联免疫吸附试验(ELISA)方法,检测临床梅毒患者血清和健康体检者正常血清,同时与梅毒螺旋体明胶凝集试验(TPPA)进行比较,根据重组蛋白与梅毒阴阳性血清的反应情况,评价重组抗原在梅毒血清学诊断中的应用价值.结果 成功构建PET-28a(+)-0993原核表达载体,经表达、纯化后获得相对分子质量约为34 000的重组蛋白;Western印迹检测显示该重组蛋白能与梅毒患者阳性血清发生特异性反应;利用纯化的TP0993重组蛋白免疫新西兰兔,能诱导新西兰兔产生特异性免疫应答.以重组蛋白为包被抗原建立间接ELISA法,对TPPA法检测的480份临床血清进行检测,与TPPA法比较ELISA法的灵敏度为88.3％,特异度为85.8％,ELISA法与TPPA法符合率为86.5％.结论 重组表达的TP0993蛋白具有较好的免疫活性,可作为梅毒血清学诊断的候选抗原之一.
目的 探討梅毒螺鏇體重組蛋白TP0993在梅毒血清學診斷中的應用價值.方法 通過生物信息學分析,穫取TP0993基因序列,構建原覈載體進行誘導錶達;Ni-NTA親閤層析柱純化重組蛋白,Western印跡檢測其免疫抗原性,用重組蛋白直接註射免疫新西蘭傢兔,評價其免疫原性.用純化的TP0993重組蛋白包被微孔闆,建立間接酶聯免疫吸附試驗(ELISA)方法,檢測臨床梅毒患者血清和健康體檢者正常血清,同時與梅毒螺鏇體明膠凝集試驗(TPPA)進行比較,根據重組蛋白與梅毒陰暘性血清的反應情況,評價重組抗原在梅毒血清學診斷中的應用價值.結果 成功構建PET-28a(+)-0993原覈錶達載體,經錶達、純化後穫得相對分子質量約為34 000的重組蛋白;Western印跡檢測顯示該重組蛋白能與梅毒患者暘性血清髮生特異性反應;利用純化的TP0993重組蛋白免疫新西蘭兔,能誘導新西蘭兔產生特異性免疫應答.以重組蛋白為包被抗原建立間接ELISA法,對TPPA法檢測的480份臨床血清進行檢測,與TPPA法比較ELISA法的靈敏度為88.3％,特異度為85.8％,ELISA法與TPPA法符閤率為86.5％.結論 重組錶達的TP0993蛋白具有較好的免疫活性,可作為梅毒血清學診斷的候選抗原之一.
목적 탐토매독라선체중조단백TP0993재매독혈청학진단중적응용개치.방법 통과생물신식학분석,획취TP0993기인서렬,구건원핵재체진행유도표체;Ni-NTA친합층석주순화중조단백,Western인적검측기면역항원성,용중조단백직접주사면역신서란가토,평개기면역원성.용순화적TP0993중조단백포피미공판,건립간접매련면역흡부시험(ELISA)방법,검측림상매독환자혈청화건강체검자정상혈청,동시여매독라선체명효응집시험(TPPA)진행비교,근거중조단백여매독음양성혈청적반응정황,평개중조항원재매독혈청학진단중적응용개치.결과 성공구건PET-28a(+)-0993원핵표체재체,경표체、순화후획득상대분자질량약위34 000적중조단백;Western인적검측현시해중조단백능여매독환자양성혈청발생특이성반응;이용순화적TP0993중조단백면역신서란토,능유도신서란토산생특이성면역응답.이중조단백위포피항원건립간접ELISA법,대TPPA법검측적480빈림상혈청진행검측,여TPPA법비교ELISA법적령민도위88.3％,특이도위85.8％,ELISA법여TPPA법부합솔위86.5％.결론 중조표체적TP0993단백구유교호적면역활성,가작위매독혈청학진단적후선항원지일.
Objective To evaluate the value of a Treponema pallidum (TP) recombinant protein TP0993 in the serodiagnosis of syphilis.Methods A bioinformatics method was used to obtain the sequence of TP0993 gene.The open reading frame (ORF) without upstream non-coding region of TP0993 gene was ligated into the expression vector PET-28a (+),which was then transformed into Escherichia coli Rosetta.Isopropyl-β-d-thiogalactoside (IPTG) was used to induce the expression of TP0993 protein.The expressed protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography.Western blot was performed to evaluate the immunoantigenicity of the protein.New Zealand rabbits were immunized with the recombinant protein for immunogenicity evaluation.Indirect enzyme linked immunosorbent assay (ELISA) was developed by using the purified recombinant protein to coat microwell plates.Anti-TP antibodies were detected by the established ELISA and TP particle agglutination assay (TPPA) in 480 clinical serum samples.Results The prokaryotic expression vector PET-28a (+)-0993 was successfully built,and a fusion protein with a relative molecular weight of about 34 000 Da was attained after IPTG-induced expression and purification.Western blot proved that the recombinant protein could specifically react with clinical sera positive for anti-TP IgG antibodies.Specific humoral response was elicited in New Zealand rabbits by the recombinant protein.Compared with TPPA,the established indirect ELISA showed a sensitivity of 88.3％ and a specificity of 85.8％.There was a consistency of 86.5％ between the indirect ELISA and TPPA.Conclusion The expressed recombinant protein showed favorable immunocompetence,and may serve as a candidate antigen for serodiagnosis of syphilis.
