结核分枝杆菌Rv3425-Rv1168c蛋白融合表达与血清学评价
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摘要
目的原核表达结核分枝杆菌Rv3425-Rv1168c融合蛋白并进行纯化,通过酶联免疫吸附试验(ELISA)方法评价重组蛋白在结核病血清学诊断中的价值。方法以结核分枝杆菌H37Rv基因组为模板,通过重叠PCR扩增得到Rv3425-Rv1168c全核酸序列克隆至表达载体pET-24b中,转入大肠杆菌BL21(DE3)进行诱导、表达和纯化,通过ELISA检测100份确诊结核病人、20例非结核呼吸疾病患者、100份健康人血清,评价融合蛋白进行临床血清学诊断的可行性。结果 Rv3425-Rv1168c融合蛋白在原核系统内获得高表达,纯化的融合蛋白经ELISA方法测定,在血清学实验中敏感性为54%,特异性为95%。结论 Rv3425-Rv1168c融合蛋白具有较高的抗原特异性和免疫原性,在结核病血清学诊断方面具有很大的应用价值。
In order to obtain fusion protein Rv3425-Rv1168c of Mycobacterium tuberculosis expression,purification in prokaryotic system and to evaluate its potential value for tuberculosis serological diagnosis,we amplified Rv3425-Rv1168c gene by overlap PCR from genome of Mycobacterium tuberculosis H37 Rv.The fragments were cloned into pET-24 bvectors.After induction,expression and purification fromE.coli BL21(DE3),the feasibility was evaluated of fusion protein for clinical serological diagnosis by ELISA assay with 100 TB patients,20 respiratory patients with a non-tuberculous and 100 healthy people sera.Rv3425-Rv1168c fusion protein was highly expressed,sensitivity and specificity were about 54%and 95% with tuberculosis serological diagnosis.Recombinant protein showed a strong antigenic ability.Results suggested Rv3425-Rv1168c protein had an important value in diagnosis of tuberculosis,and might be selected as one of diagnostic antigens of tuberculosis.
In order to obtain fusion protein Rv3425-Rv1168c of Mycobacterium tuberculosis expression,purification in prokaryotic system and to evaluate its potential value for tuberculosis serological diagnosis,we amplified Rv3425-Rv1168c gene by overlap PCR from genome of Mycobacterium tuberculosis H37 Rv.The fragments were cloned into pET-24 bvectors.After induction,expression and purification fromE.coli BL21(DE3),the feasibility was evaluated of fusion protein for clinical serological diagnosis by ELISA assay with 100 TB patients,20 respiratory patients with a non-tuberculous and 100 healthy people sera.Rv3425-Rv1168c fusion protein was highly expressed,sensitivity and specificity were about 54%and 95% with tuberculosis serological diagnosis.Recombinant protein showed a strong antigenic ability.Results suggested Rv3425-Rv1168c protein had an important value in diagnosis of tuberculosis,and might be selected as one of diagnostic antigens of tuberculosis.
引文
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[17]孙卫国,李邦印,王倩,等.重组结核分枝杆菌PPE17蛋白原核可溶性表达纯化和血清学诊断研究[J].中国预防医学杂志,2011,12(11):910-913.
[2]陈秀平.超声对肾结核及其分型的诊断价值和意义[J].实用临床医学,2016,17(5):68-69.DOI:10.13764/j.cnki.lcsy.2016.05.026
[3]陈慧姮,薛如君,田歆,等.长期误诊的疣状皮肤结核1例[J].中国皮肤性病学杂志,2017,31(8):925-926.DOI:10.19367/j.cnki.1000-2707.2015.03.036
[4]曹仕鹏,蒋之,傅满姣.脊柱结核延误诊断及治疗困难原因分析[J].临床误诊误治,2015,28(12):1-4.DOI:10.3969/j.issn.1002-3429.2015.12.001
[5]Bello AK,Njoku CH.Tuberculosis:current trends in diagnosis and treatment[J].Niger J Clin Pract,2005,8(2):118-124.
[6]Zhang H,Wang J,Lei J,et al.PPE protein(Rv3425)from DNA segment RD11of Mycobacterium tuberculosis:apotential B-cell antigen used for serological diagnosis to distinguish vaccinated controls from tuberculosis patients[J].Clin Microbiol Infect,2007,13(2):139-145.DOI:10.1111/j.1469-0691.2006.01561.x
[7]王倩,罗微,屈子璐,等.结核分枝杆菌Rv3425蛋白抗原免疫学特性的研究[J].中国免疫学杂志,2017,33(1):31-35.DOI:10.3969/j.issn.1000-484X.2017.01.006
[8]Abdallah AM,Verboom T,Hannes F,et al.A special secretion system mediates PPE41transportin pathogenic mycobacteria[J].Molecular Microbiology,2006,62(3):667-679.DOI:10.1111/j.1365-2958.2006.05409.x
[9]余晓丽,孙战强,周晨俊,等.结核分枝杆菌Rv1168c蛋白的基因表达、纯化及结构分析[J].微生物学报,2010,50(7):931-936.DOI:10.13343/j.cnki.wsxb.2010.07.019
[10]冯英凯,王勇,黄正谷,等.蛋白芯片技术与其他4种检测方法对结核病诊断价值的比较[J].中华肺部疾病杂志,2017,10(4):427-430.DOI:10.3877/cma.j.issn.1674-6902.2017.04.012
[11]宁博.结核分枝杆菌PCR检测对菌阴肺结核的诊断价值研究[J].中国医学工程,2015,23(12):69.
[12]蒋瑜,吴婷.结核感染T细胞斑点试验及抗结核抗体检测在结核诊断中的应用价值评价[J].实用预防医学,2017,38(16):2234-2236.DOI:10.3969/j.issn.1673-4130.2017.16.022
[13]叶伟南,莫荣新,李少芳.三种方法检测结核分枝杆菌复合群的比较分析[J].临床肺科杂志,2017,22(10):1921-1923.DOI:10.3969/j.issn.1009-6663.2017.010.047
[14]Choudhary RK,Mukhopadhyay S,Chakhaiyar P,et al.PPEantigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response[J].Infect Immun,2003,71(8):6338-6343.DOI:10.1128/IAI.71.11.6338-6343.2003
[15]Zhang SL,Zhao JW,Sun ZQ,et al.Development and evaluation of a novel multipleantigen ELISA for serodiagnosis of Tuberculosis[J].Tuberculosis,2009,89(4):278-284.DOI:10.1016/j.tube.2009.05.005
[16]孙卫国,张灵霞,熊志红,等.结核分枝杆菌Rv3425蛋白原核可溶性表达[J].实用预防医学,2014,21(12):1409-1411.DOI:10.3969/j.issn.1006-3110.2014.012.001
[17]孙卫国,李邦印,王倩,等.重组结核分枝杆菌PPE17蛋白原核可溶性表达纯化和血清学诊断研究[J].中国预防医学杂志,2011,12(11):910-913.