中分子量神经丝亚单位单克隆抗体的制备及初步应用

发布时间：2018-03-13 18:33

本文选题：神经丝M　切入点：单克隆抗体　出处：《山东大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：制备抗神经丝中分子量亚单位(NF-M)的单克隆抗体(McAb),为神经丝检测方法的建立奠定基础。方法：将人、大鼠、小鼠NF-M氨基酸序列进行比对,选择合适的氨基酸序列GNPSAYRRVTETRSSFSRVSGSPSSGFRSQSWSRGSP-C进行多肽合成,合成后的多肽通过Sulfo-SMCC与匙孔槭血蓝蛋白(KLH)载体蛋白偶联。将偶联后的多肽KLH-NF-M作为抗原,以腹腔注射的方式免疫BALB/c小鼠。首次免疫使用福氏完全佐剂乳化抗原,其后的免疫使用福氏不完全佐剂乳化,每隔2周免疫一次。多次免疫后,小鼠内眦取血,离心取血清。以NF-M多肽包被酶标板,用酶联免疫吸附试验(ELISA)测定小鼠外周血中抗体效价。选择血清抗体效价高的小鼠,进行脾内免疫,3天后处死小鼠,取其脾细胞。采用细胞融合技术,PEG4000为融合剂,将小鼠脾细胞和小鼠骨髓瘤细胞(sp2/0细胞)融合。使用HAT培养基筛选杂交瘤细胞。ELISA检测杂交瘤细胞培养上清中McAb,对分泌抗NF-M的McAb的细胞株以有限稀释法进行克隆化。获得能稳定分泌单克隆抗体的杂交瘤细胞株。以竞争抑制,免疫学方法对该McAb亚类进行鉴定。以NF-M多肽为竞争抗原应用竞争抑制试验检测McAb的特异性。将正常大鼠大脑组织匀浆后上样,进行SDS-PAGE电泳,电转移至PVDF膜,以杂交瘤细胞产生的抗体为一抗,进行Western blot试验,检测抗体与大脑组织中的NF-M特异性结合的能力。以正常大鼠大脑作石蜡切片,以杂交瘤细胞产生的抗体为一抗,进行免疫组化试验,进一步鉴定该单克隆抗体的特异性。结果：BALB/c小鼠经腹腔免疫4次后,ELISA检测血清抗体效价为1：512000。经细胞融合后,融合率为46.61%, ELISA检测抗体阳性率7.26%。对分泌抗NF-M的阳性细胞株有限稀释法克隆化3-5次后,单克隆孔抗体分泌阳性率达到100%,建立了一株能稳定分泌抗NF-M的杂交瘤细胞株2C1。经单克隆抗体亚类鉴定2C1所产生的抗体为IgG1。多肽竞争抑制结果：竞争抑制曲线为y=0.4127x+0.2691,相关系数r=0.9912,证明McAb是针对NF-M的。Western blot实验在160KD分子量附近可见一条清楚的蛋白条带,与NF-M分子量大小相符。表明该McAb与正常大鼠大脑匀浆中的NF-M蛋白有较好的特异性反应。正常大鼠大脑及脊髓石蜡切片的免疫组化结果显示,神经元胞核周围棕褐色着色颗粒,表明该McAb与正常大鼠大脑切片组织中的NF-M有特异性反应。结论：制备了特异性抗NF-M的McAb,该McAb在Western blot和免疫组化实验中均与大鼠NF-M特异性结合,可用于NF-M诊断试剂的研制。
[Abstract]:Aim: to prepare monoclonal antibody against NF-M in neurofilament, and to lay a foundation for the establishment of a method for the detection of neurofilament. Methods: the amino acid sequences of human, rat and mouse NF-M were compared, and the suitable amino acid sequence GNPSAYRRVTETRSSFSRVSGSPSSGFRSQSWSRGSP-C was selected for peptide synthesis. The synthesized polypeptide was coupled with the carrier protein of Acer mono hemocyanin (KLH) by Sulfo-SMCC. KLH-NF-M was used as antigen to immunize BALB/c mice by intraperitoneal injection. Freund's complete adjuvant emulsified antigen was used for the first time. After repeated immunization, the blood was collected from the inner canthus of the mice, and the serum was collected by centrifugation. The enzyme labeled plate was coated with NF-M polypeptide, and then the adjuvant was emulsified by Freund's incomplete adjuvant. Enzyme linked immunosorbent assay (Elisa) was used to determine the antibody titers in peripheral blood of mice. Mice with high antibody titer were selected. The mice were killed after 3 days of intrasplenic immunization, and the spleen cells were taken out. PEG4000 was used as fusion agent. HAT medium was used to screen hybridoma cells. Elisa was used to detect McAbs in the culture supernatant of hybridoma cells. The cell lines secreting McAb against NF-M were cloned by limited dilution method. To obtain hybridoma cell lines which can secrete monoclonal antibody stably. The subclass of McAb was identified by immunological method. The specificity of McAb was detected by competitive inhibition test with NF-M polypeptide as competitive antigen. After homogenate of normal rat brain tissue was sampled, SDS-PAGE electrophoresis was performed, and then transferred to PVDF membrane. The Western blot test was carried out to detect the ability of the antibody to bind to the specific NF-M in the brain tissue. The normal rat brain was used as paraffin section, and the antibody produced by the hybridoma cell was used as the first antibody. The specificity of the monoclonal antibody was further identified by immunohistochemistry. Results after 4 times of intraperitoneal immunization, the titer of serum antibody detected by Elisa was 1: 512000. After cell fusion, the fusion rate was 46.61.The positive rate of ELISA antibody was 7.26.The antibody was cloned by limited dilution method for 3-5 times. A hybridoma cell line 2C1, which can stably secrete anti NF-M, was established. The antibody produced by 2C1 was identified as IgG1 by monoclonal antibody subclass. The result of peptide competition inhibition: the competitive inhibition curve was 0.4127x. The correlation coefficient is 0.2691 and the correlation coefficient is 0.9912. It is proved that McAb is a clear protein band near the molecular weight of 160KD. The molecular weight of McAb was consistent with the molecular weight of NF-M. It showed that the McAb had a good specific reaction with the NF-M protein in the brain homogenate of normal rats. The immunohistochemical results of paraffin sections of brain and spinal cord of normal rats showed that the brown stained granules around the nucleus of neurons were brown. The results showed that the McAb had a specific reaction with NF-M in normal rat brain tissue. Conclusion: McAbs with specific anti NF-M have been prepared. The McAb binds specifically to rat NF-M in Western blot and immunohistochemistry experiments, and can be used in the development of NF-M diagnostic reagent.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【相似文献】