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抗人抗苗勒管激素N端439～451表位单克隆抗体的制备及其性能研究

Monoclonal Antibody Against N Terminal 439-451 Epitopes of Anti-Mullerian Hormone and Its Properties

    摘要
  • 摘要:
      目的  制备抗人抗苗勒管激素（anti-mullerian hormone，AMH）N端特定表位肽的特异单克隆抗体并对其特性进行鉴定。
      方法  利用生物信息分析软件预测AMH特定多肽片段，合成AMH成熟N端区域的4个抗原性好的表位肽段作为筛选靶抗原。用重组AMH蛋白抗原免疫BALB/c小鼠，取小鼠脾细胞与SP/20细胞进行细胞融合，通过杂交瘤技术制备抗AMH单克隆抗体，用N端4个表位肽（表位1：439～451 RGRDPRGPGRAQR，表位2：273-285 PPRPSAELEESPP，表位3：42～54 DLDWPPGSPQEPL，表位4：494～506 WPQSDRNPRYGNH）分别作抗原，间接ELISA和Western blot对筛选获得的特异抗表位单抗的抗原结合特性进行鉴定。
      结果  筛选到两株能够稳定分泌抗人AMH表位1（anti-AMH-1）和表位2（anti-AMH-2）的单克隆抗体杂交瘤细胞株，其分泌的单抗分别命名为anti-AMH-1和anti-AMH-2。该2株单抗细胞表达纯化后，检测其抗体效价分别为1∶12 000和1∶1 600。Western blot确认两种单抗除效价有差异外，识别抗原也有差异，anti-AMH-1不但能识别AMH的N端439-451表位肽，而且能识别重组AMH的氨基酸序列（rAMH），也能识别卵巢组织。anti-AMH-2能识别rAMH和卵巢组织。
      结论  成功构建了抗人AMH N端的特异表位的2种单克隆抗体。

     

    Abstract:
      Objective  To prepare the specific monoclonal antibody against the N-terminal specific epitope peptide of anti-mullerian hormone (AMH) and to identify its specificity.
      Methods  Using bioinformatics analysis software to predict the specific peptide fragment of AMH. Then synthesized four antigenic epitope peptide segments of mature N-terminal region of AMH as the screening target antigen. Synthesized AMH wholegene.Using the prokaryotic expression system to abtain recombinant AMH protein. Immunized BALB/c mice with the recombinant AMH, and prepared mouse spleen cells for fusing with SP/20 cells. Preparation of AMH monoclonal antibody by hybridoma technology. The monoclonal antibodies against AMH were screened by using four N-terminal epitope peptides (1: 439-451 RGRDPRGPGRAQ, 2: 273-285 PPRPSAELEESPP, 3: 42-54 DLDWPPGSPQEPL, 4: 494-506 WPQSDRNPRYGNH) as antigens, and indirect ELISA and Western blot were used to identify the antigen binding characteristics of the selected monoclonal antibodies.
      Results  Two hybridoma cell lines with stable anti-AMH-1 and anti-AMH-2 antibody activities were screened. The two antibodies were named anti-AMH-1 and anti-AMH-2 respectively. The antibody titers were 1∶12 000 and 1∶1 600 after purification. Western blot confirmed that the two McAbs recognized different antigens. Anti-AMH-1 could not only recognize the N-terminal 439-451 epitope peptide of AMH, but also recognize the amino acid sequence of recombinant AMH, as well as the ovarian tissue. Anti-AMH-2 could recognize recombinant AMH and ovarian tissue.
      Conclusion  Two monoclonal antibodies against N-terminal specific epitopes of human AMH were successfully constructed.

     

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