妊娠相关血浆蛋白A的纯化和单克隆抗体的制备

发布时间：2018-10-22 10:07

【摘要】： 背景：妊娠目关血浆蛋白A(Pregnancy-associated Plasma Protein A，，PAPP-A)是一种与妊娠相关的大分子糖蛋白，主要是由胎盘合体滋养层和蜕膜产生。母体血清PAPP-A含量与妊娠周期密切相关，随孕周的增加而持续上升，到妊娠末期达到高峰。PAPP-A是早孕阶段母血中一个敏感特异的血清标志物，孕早期母血中低水平的PAPP-A与后来发生的各种染色体异常、胎儿畸形、产科并发症等异常妊娠有关。目前，在早孕阶段测定孕妇血清中PAPP-A已经应用于临床异常妊娠的预测，特别对于唐氏综合征等染色体异常的筛查有较大的临床价值。由于母血中低水平的PAPP-A与胎盘功能异常有关，因此PAPP-A是早孕阶段预测异常妊娠的一个有价值的指标。新近的研究表明PAPP-A是急性冠脉综合征早期识别、进行危险分层以及评价预后的一个新标志物。目的：从健康孕妇血清中分离纯化PAPP-A蛋白，用所纯化的抗原免疫小鼠，筛选分泌PAPP-A单克隆抗体的杂交瘤细胞株，制备抗人PAPP-A单克隆抗体，为建立PAPP-A检测方法及其临床应用提供基础。方法：利用DEAE-Sepharose CL-6B离子交换和Heparin-Sepharose CL-6B亲和层析从晚孕血清中分离纯化PAPP-A，变性SDS-PAGE测定其相对分子质量为200 kDa，western blotting鉴定其特异性。取24μg纯化后的PAPP-A加等体积福氏佐剂免疫Balb／c小鼠，共3次，融合前3天加强免疫1次，用聚乙二醇(PEG)行细胞融合，用杂交瘤细胞选择性培养基次黄嘌呤、氨基喋呤、胸腺嘧啶核苷(HAT)及次黄嘌呤、胸腺嘧啶核苷(HT)选择性培养，间接酶联免疫吸附实验(ELISA)筛选培养上清，包被的抗原为纯化后的PAPP-A，选择对PAPP-A阳性的多克隆孔进行亚克隆，间接ELISA筛选亚克隆板孔的细胞培养上清。扩大培养分泌PAPP-A单克隆抗体的细胞株，以5×10~5-1×10~6个细胞／只接种于一周前腹腔注射0.5ml／只液体石蜡油的Balb／c小鼠。接种后7天开始收集腹水，间接ELISA检测腹水PAPP-A抗体滴度，所得腹水用蛋白A亲和层析柱纯化单抗，对得到单克隆抗体进行分析鉴定。结果：纯化的PAPP-A蛋白行SDS-PAGE电泳显示主条带为PAPP-A亚基的位置，其分子量大小约200 kDa，proMBP亚基的分子量大小约50-90 kDa。PAPP-A亚基可与PAPP-A单克隆抗体发生特异性反应。筛选出12株稳定分泌PAPP-A McAbs的杂交瘤细胞株，8株是IgG1亚型，4株是IgG2a亚型。单抗SDS-PAGE分析显示轻链分子量约为28.7kDa，重链约为51.3 kDa。Western blotting和免疫组织化学分析证实所获得的McAb对PAPP-A有较高的特异性及敏感性。结论：从孕妇血清中成功分离得到妊娠血浆相关蛋白A，并获得稳定分泌抗人PAPP-A单克隆抗体的细胞株，制备出抗人PAPP-A的单克隆抗体。可用于PAPP-A免疫检测方法的建立。
[Abstract]:Background: Pregnancy-associated Plasma Protein PAPP-A is a macromolecular glycoprotein associated with pregnancy, mainly produced by placenta syncytiotrophoblast and decidua. The maternal serum PAPP-A content is closely related to the pregnancy cycle and increases continuously with the increase of gestational week, and reaches its peak at the end of pregnancy. PAPP-A is a sensitive and specific serum marker in maternal blood during early pregnancy. The low level of PAPP-A in maternal blood in early pregnancy is related to the subsequent abnormal pregnancy, such as chromosomal abnormalities, fetal malformations, obstetric complications and so on. At present, the determination of serum PAPP-A in pregnant women during early pregnancy has been applied to predict clinical abnormal pregnancy, especially for the screening of chromosomal abnormalities such as Down's syndrome. Because the low level of PAPP-A in maternal blood is associated with abnormal placental function, PAPP-A is a valuable index to predict abnormal pregnancy in early pregnancy. Recent studies have shown that PAPP-A is a new marker for early identification, risk stratification and prognostic evaluation of acute coronary syndrome. Objective: to isolate and purify PAPP-A protein from the serum of healthy pregnant women, immunize mice with purified antigen, screen hybridoma cell lines secreting monoclonal antibodies against PAPP-A, and prepare monoclonal antibodies against human PAPP-A. To provide a basis for the establishment of PAPP-A detection method and its clinical application. Methods: DEAE-Sepharose CL-6B ion exchange and Heparin-Sepharose CL-6B affinity chromatography were used to isolate and purify PAPP-A, denatured SDS-PAGE from late pregnancy serum. The relative molecular weight of PAPP-A, denatured SDS-PAGE was 200 kDa,western blotting to identify its specificity. Balb/c mice were immunized with 24 渭 g purified PAPP-A and equal volume Freund's adjuvant for 3 times. The mice were immunized once 3 days before fusion. The cells were fused with polyethylene glycol (PEG). Hypoxanthine and aminopterin were used as the selective culture medium of hybridoma cells. Thymidine (HAT), Hypoxanthine and thymine nucleoside (HT) were selectively cultured. Indirect enzyme-linked immunosorbent assay (ELISA) was used to screen the supernatant of the culture supernatant. The coated antigen was the purified PAPP-A, to subclone the PAPP-A positive polyclonal pore. Indirect ELISA was used to screen the supernatant of cell culture. The cell lines secreting monoclonal antibody to PAPP-A were expanded and inoculated with 5 脳 10 ~ (-1) 脳 10 ~ (6) cells / Balb/c mice injected intraperitoneally with 0.5ml/ liquid paraffin one week ago. The ascites were collected 7 days after inoculation. The titer of PAPP-A antibody in ascites was detected by indirect ELISA. The monoclonal antibody was purified by protein A affinity chromatography and the monoclonal antibody was analyzed and identified. Results: the purified PAPP-A protein was identified as the PAPP-A subunit by SDS-PAGE electrophoresis. The molecular weight of its molecular weight was about 200 kDa,proMBP and the molecular weight of the kDa,proMBP subunit was about 50-90 kDa.PAPP-A, which could react specifically with the PAPP-A monoclonal antibody. Twelve hybridoma cell lines stably secreting PAPP-A McAbs were screened, 8 of them were IgG1 subtypes and 4 were IgG2a subtypes. SDS-PAGE analysis showed that the molecular weight of the light chain was about 28.7 kDa, and the heavy chain was about 51.3 kDa.Western blotting. The obtained McAb was confirmed to be specific and sensitive to PAPP-A by immunohistochemical analysis. Conclusion: pregnancy plasma associated protein A was isolated from maternal serum and the cell lines secreting monoclonal antibodies against human PAPP-A were obtained. Monoclonal antibodies against human PAPP-A were prepared. It can be used for the establishment of PAPP-A immunoassay.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R714;R392

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