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
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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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