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Purification of Antigen 85 Complex of Mycobacterium tuberculosis
结核分枝杆菌抗原85复合物的纯化   

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Abstract

Serodiagnosis of tuberculosis using purified native antigens is one of the approaches for the early detection of TB. The Antigen 85 complex consisting of Ag 85A, 85B and 85C are the most abundant antigens in the culture supernatant of mycobacteria. It has also been shown to be the immunodominant (the property of an antigenic determinant that causes it to be responsible for the major immune response in a host) antigens of CFA (culture filtrate antigens – proteins secreted by mycobacteria into the culture medium). This protocol gives the details for purification of the Ag85 complex from CFA and also the method of isolation of the individual components of the complex by column chromatography.

Materials and Reagents

  1. NaH2PO4
  2. EDTA
  3. DTT
  4. Tris
  5. NaCl
  6. Glycine
  7. Ethylene glycol
  8. HCI
  9. QAE Sepharose Column Starting buffer (see Recipes)
  10. QAE Sepharose Column Elution buffer (see Recipes)
  11. Buffer A (see Recipes)
  12. Buffer B (see Recipes)
  13. Buffer C (see Recipes)

Equipment

  1. High Performance Liquid Chromatography (HPLC) system (Millenium V2.00, Waters, USA)
  2. SDS-PAGE (Mini-PROTEAN® Tetra Cell, catalog number: 165-8000 )
  3. Phenyl Sepharose HP column (3.5 x 1 cm) (GE Healthcare, catalog number: 17-1082-01 )
  4. QAE sepharose column (Amersham Pharmacia – anion exchange chromatography).

Procedure

  1. Purification of 30 kDa antigen:
    The secreted Antigen 85 complex of mycobacteria consists of three proteins Ag 85A, B and C. Among these Antigen 85B otherwise known as 30 kDa antigen is one of the major immunogenic antigen.
    The 30 kDa antigen was purified from the whole culture filtrate of M. tuberculosis by first passing through QAE column followed by passing the 30 kDa enriched fraction through a Phenyl Sepharose hydrophobic interaction column.
    1. Anion exchange chromatography-QAE sepharose (purification of Ag85 complex)
      (PURIFICATION OF AG 85 COMPLEX)
      1. Culture filtrate antigen (CFA) between 10 to 30 mg was prepared as mentioned in the 38 kDa antigen purification protocol (Hyperlink here) was separated into multiple fractions by passing through a prepacked anion exchange column of QAE Sepharose. 0.05 M Tris-HCI buffer (pH 8.1) was used as the starting buffer and 0.05 M Tris-HCI, pH 8.1, with 1.0 M sodium chloride was used as the elution buffer. The separation was carried out in the High Performance Liquid Chromatography (HPLC, Millenium V2.00,  Waters, USA) system under standard running conditions. The eluted fractions were analyzed on 12.5% SDS-PAGE which separates proteins based on its molecular weight, was run under standard conditions (120 V, 1.5 A). The Ag85 complex is identified as a 30/31 kDa doublet band on the gel. The fractions containing Antigen 85 complex were pooled and concentrated by Amicon centrifuge filters with the molecular cut off of 3 kDa.
      2. Further purification of the A, B and C components from the antigen 85 complex was done by passing it through the hydrophobic interaction chromatography (HIC) column namely the Phenyl Sepharose HP column.

    2. Hydrophobic interaction chromatography-phenyl sepharose (separation of the individual components of the antigen 85 complex)
      1. Purification of the antigen 85 complex into its individual components Ag 85A; 85B and 85C was done by passing it through the Phenyl Sepharose HP column using the Biologic Chromatography system (FPLC, Bio-Rad, USA). Three different buffers were used during the run. The buffers A, B, C used are mentioned under recipes section.
      2. The run conditions were such that the column was subjected to an isocratic flow of Buffer A for 10 min (flow rate 1 ml/min), followed by the injection of the sample (2 ml) which was dialysed against buffer A at the rate of 0.5 ml/min. Then an isocratic flow of Buffer B for 10 min at the rate of 1 ml/min was used for washing the unbound material.
      3. Elution of the individual components of Antigen 85 complex namely Ag85A, B and C were carried out with a linear gradient of Buffer C (from 0 -100%) for 30 min with the flow rate of 1 ml/min. Subsequently, the protein containing fractions were identified by optical density measured at 280 nm on a spectrophotometer. The fractions containing Ag 85B were pooled, concentrated and checked on 12.5% SDS PAGE for purity.

Recipes

  1. QAE Sepharose Column Starting buffer
    0.05 M Tris-HCI (pH 8.1)
  2. QAE Sepharose Column Elution buffer
    0.05 M Tris-HCI (pH 8.1)
    1 M NaCl
  3. Buffer A
    0.01 M NaH2PO4
    1 mM EDTA
    1 mM DTT (pH 6.8)
  4. Buffer B
    0.01 M Tris
    100 mM Glycine
    1 mM EDTA
    1 mM OTT(pH 8.9)
  5. Buffer C
    0.01 M Tris
    100 mM Glycine
    1 mM DTT
    50% (v/v) Ethylene Glycol (pH 8.9)

Acknowledgments

This protocol was extracted from the original publication by Devi et al. (2002). This work was supported by Indo US VAP & DBT India granted for Dr. Alamelu Raja.

References

  1. Devi, K. R., Kumar, K. S., Ramalingam, B. and Alamelu, R. (2002). Purification and characterization of three immunodominant proteins (38, 30, and 16 kDa) of Mycobacterium tuberculosis. Protein Expr Purif  24(2): 188-195.
  2. Raja, A., Uma Devi, K. R., Ramalingam, B. and Brennan, P. J. (2004). Improved diagnosis of pulmonary tuberculosis by detection of free and immune complex-bound anti-30 kDa antibodies. Diagn Microbiol Infect Dis 50(4): 253-259.

简介

使用纯化的天然抗原的结核病的血清诊断是早期检测TB的方法之一。 由Ag 85A,85B和85C组成的抗原85复合物是分枝杆菌培养物上清液中最丰富的抗原。 它也被证明是CFA(培养物滤液抗原 - 分枝杆菌分泌到培养基中的蛋白质)抗原的免疫显性(导致其负责宿主主要免疫应答的抗原决定簇的性质)。 该方案给出了从CFA纯化Ag85复合物的细节以及通过柱色谱法分离复合物的各个组分的方法。

材料和试剂

  1. NaH 2 PO 4 sub
  2. EDTA
  3. DTT
  4. Tris
  5. NaCl
  6. 甘氨酸
  7. 乙二醇
  8. HCl
  9. QAE琼脂糖柱起始缓冲液(见配方)
  10. QAE Sepharose柱洗脱缓冲液(见配方)
  11. 缓冲液A(参见配方)
  12. 缓冲液B(参见配方)
  13. 缓冲区C(参见配方)

设备

  1. 高效液相色谱(HPLC)系统(Millenium V2.00,Waters,USA)
  2. SDS-PAGE(Mini-PROTEAN Tetra Cell,目录号:165-8000)
  3. 苯基琼脂糖HP柱(3.5×1cm)(GE Healthcare,目录号:17-1082-01)
  4. QAE琼脂糖柱(Amersham Pharmacia - 阴离子交换层析)。

程序

  1. 30 kDa抗原的纯化:
    分枝杆菌的分泌的抗原85复合物由三种蛋白质Ag 85A,B和C组成。其中这些抗原85B或称为30kDa抗原是主要免疫原性抗原之一。
    通过首先通过QAE柱,随后使30kDa富集的级分通过苯基琼脂糖疏水相互作用柱,从结核分枝杆菌的全部培养物滤液中纯化30kDa抗原 。
    1. 阴离子交换色谱-QAE琼脂糖(纯化Ag85复合物)
      (纯度85复合)
      1. 通过通过QAE Sepharose的预填充阴离子交换柱将在38kDa抗原纯化方案(在此Hyperlink)中提及的10至30mg的培养物滤液抗原(CFA)分离成多个级分。使用0.05M Tris-HCl缓冲液(pH 8.1)作为起始缓冲液,使用0.05M Tris-HCl,pH 8.1,用1.0M氯化钠作为洗脱缓冲液。在标准运行条件下在高效液相色谱(HPLC,Millenium V2.00,Waters,USA)系统中进行分离。在12.5%SDS-PAGE上分析洗脱的级分,其在标准条件(120V,1.5A)下运行基于其分子量分离蛋白质。 Ag85复合物被鉴定为凝胶上的30/31kDa双峰带。合并含有抗原85复合物的级分,并通过分子量截止为3kDa的Amicon离心过滤器浓缩。
      2. 通过使其通过疏水作用层析(HIC)柱(即Phenyl Sepharose HP柱),进行来自抗原85复合物的A,B和C组分的进一步纯化。

    2. 疏水相互作用层析 - 苯基琼脂糖(分离抗原85复合物的单个组分)
      1. 将抗原85复合物纯化成其各个组分Ag 85A; 85B和85C通过使用生物色谱系统(FPLC,Bio-Rad,USA)使其通过Phenyl Sepharose HP柱来进行。运行期间使用三种不同的缓冲液。使用的缓冲区A,B,C在配方部分下提及
      2. 运行条件是使柱经受缓冲液A的等度流动10分钟(流速1ml/min),然后注入样品(2ml),其以缓冲液A的速率 0.5ml/min。 然后使用以1ml/min的速率的缓冲液B的等度流动10分钟来洗涤未结合的物质。
      3. 抗原85复合物(即Ag85A,B和C)的各个组分的洗脱用缓冲液C的线性梯度(从0-100%)进行30分钟,流速为1ml/min。 随后,通过在分光光度计上在280nm测量的光密度鉴定含蛋白质的级分。 合并含Ag 85B的级分,浓缩并在12.5%SDS PAGE上检查纯度。

食谱

  1. QAE Sepharose色谱柱起始缓冲液
    0.05M Tris-HCl(pH8.1)
  2. QAE Sepharose色谱柱洗脱缓冲液
    0.05M Tris-HCl(pH8.1) 1 M NaCl
  3. 缓冲区A
    0.01M NaH 2 PO 4 sub/
    1mM DTT(pH 6.8)
  4. 缓冲区B
    0.01 M Tris
    100 mM甘氨酸 1mM EDTA
    1mM OTT(pH8.9)
  5. 缓冲区C
    0.01 M Tris
    100 mM甘氨酸 1 mM DTT
    50%(v/v)乙二醇(pH8.9)

致谢

该方案从Devi等人的原始出版物中提取((2002)。 这项工作得到了Indo US VAP& DBT印度授予Dr. Alamelu Raja博士。

参考文献

  1. Devi,K.R.,Kumar,K.S.,Ramalingam,B。和Alamelu,R。(2002)。 分枝杆菌的三种免疫显性蛋白(38,30和16 kDa)的纯化和表征 肺结核 。 Protein Expr Purif 24(2):188-195。
  2. Raja,A.,Uma Devi,KR,Ramalingam,B.and Brennan,PJ(2004)。通过检测游离和免疫复合物结合的抗30kDa抗体来改善肺结核的诊断。 Diagn Microbiol Infect Dis 50(4):253-259。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ranganathan, U. D. and Raja, A. (2013). Purification of Antigen 85 Complex of Mycobacterium tuberculosis. Bio-protocol 3(3): e324. DOI: 10.21769/BioProtoc.324.
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