人妊娠特异性β1糖蛋白胶体金检测试纸条的研制

发布时间：2018-06-24 09:19

本文选题：妊娠特异性β1糖蛋白 + 单克隆抗体　；参考：《吉林大学》2015年硕士论文

【摘要】：妊娠特异性β1糖蛋白（Pregnancy Specific β1Glycoprotein, PSG1）是妊娠期间由胎盘滋养层细胞合成的一种蛋白质，合成后外分泌到母体外周血液循环中。在母体血清中PSG1低于正常值的情况下，常常造成一些不良后果，如胎儿发育不良及流产等[1]。同样病理状态下，PSG1也被发现存在于许多肿瘤组织中。作为一种妊娠的特异性糖蛋白，PSG1具有监测胎盘功能、检测胎儿生长受限、检测肿瘤细胞、参与免疫调节、以及维持妊娠期间母体的免疫耐受环境等功能[2]。一般认为,在早孕的诊断、胎盘功能的监测、以及胎儿预后的判定等一些应用上，PSG1蛋白含量的高低可以作为结果判断的一个依据。因此对PSG1的研究也越来越受到重视。先前工作中，实验室已经构建了能稳定表达PSG1单抗的杂交瘤细胞株、重组蛋白的原核表达系统。在此基础上，从中选择两种抗原表面结合位点之间影响小的细胞株。采用双抗体夹心法的原理，以建立一种灵敏特异、快速简便的PSG1检测方法。 1. PSG1重组蛋白的表达和纯化复苏具有原核表达质粒的菌株DE3，并扩大培养，制备包涵体蛋白。复性、纯化后，用于后续的检测。纯化后蛋白的浓度为0.6mg/ml。 2. PSG1单抗细胞株的筛选。复苏3株单抗细胞株，并对其稳定性进行检测。用叠加法ELISA实验对实验室制备的3株单抗细胞株进行筛选，挑选出抗原识别表位影响小的两株单抗9G6和2D10。 3.单克隆抗体的制备以及纯化。取健康小鼠，注射杂交瘤细胞在腹腔中。取腹水来制备单抗，离心后取上清，，除去杂质以及表面脂质。然后用G蛋白进一步纯化抗体。SDS-PAGE电泳及WB检测纯化效果。测定其蛋白浓度2D10和9G6分别为1.69mg/ml，1.37mg/ml。ELISA检测抗体的效价大于1:2.48×106。 4.制备胶体金，确定标记条件及组装条件。制备胶体金。通过检测其吸收光波长，判断其直径在30nm左右。采用双抗体夹心法，9G6作为标记抗体，2D10作为捕获抗体，包被于硝酸纤维素膜上。兔抗小鼠IgG抗体固定在C线上。并确定了蛋白的标记条件以及组装条件。 5.试纸条的性能评价。组装后，通过对重组蛋白的检测，判断其灵敏度较好。只是在检测天然蛋白时，其灵敏度的降低。检测与胎盘分泌的其他蛋白，可以看出试纸条有较好的特异性。检测不同温度下保存的试纸条，其稳定性良好。本研究获得了活性较好，纯度较高的重组PSG1蛋白以及单克隆抗体，制备了具有良好灵敏度，稳定性以及特异性的PSG1胶体金检测试纸条。下一步需要做的是将检测试纸条定量化，进一步优化条件，使之与天然PSG1蛋白有更加明显的反应，以满足临床检测的需要。
[Abstract]:Pregnancy specific 尾 1 glycoprotein (PSG1) is a kind of protein synthesized by placental trophoblastic cells during pregnancy, which is secreted into maternal peripheral blood circulation. When PSG1 in maternal serum is lower than normal, it often causes some adverse consequences, such as fetal dysplasia and abortion. In the same pathological state, PSG1 is also found in many tumor tissues. As a kind of gestational specific glycoprotein, PSG1 has the functions of monitoring placenta, detecting fetal growth restriction, detecting tumor cells, participating in immune regulation, and maintaining maternal immune tolerance environment during pregnancy [2]. It is generally believed that the level of PSG1 protein can be used as a basis for judging the results in the diagnosis of early pregnancy, the monitoring of placental function and the determination of fetal prognosis. Therefore, more and more attention has been paid to the study of PSG1. In previous work, the prokaryotic expression system of recombinant protein was constructed in a hybridoma cell line which could stably express PSG1 monoclonal antibody. On this basis, two kinds of antigen surface binding sites were selected to affect the small cell lines. The principle of double antibody sandwich method was used to establish a sensitive, specific, rapid and simple method for the detection of PSG1. 1. Expression and purification of PSG1 Recombinant protein and purification of strain DE3 with prokaryotic expression plasmid and expanded culture to prepare inclusion body protein. Renaturation, purification, and subsequent detection. The concentration of purified protein was 0.6 mg / ml. 2. Screening of PSG1 McAb Cell Line. Three McAb cell lines were resuscitated and their stability was tested. Three McAb cell lines prepared in laboratory were screened by superposition Elisa. Two McAbs 9G6 and 2D103which had little effect on antigen recognition epitope were selected. Preparation and purification of monoclonal antibodies. Healthy mice were injected with hybridoma cells in abdominal cavity. Ascites were used to prepare McAbs, centrifuged and supernatant to remove impurities and surface lipids. Then the antibody was further purified by G protein. SDS-PAGE electrophoresis and WB were used to detect the purification effect. The antibody titers of 2D10 and 9G6 were 1.69 mg / ml, 1.37 mg / ml and 1.37 mg / ml, respectively. The titer of Elisa was more than 1: 2.48 脳 106.4. Colloidal gold was prepared, labeling conditions and assembly conditions were determined. Preparation of colloidal gold. By measuring the wavelength of the absorbed light, the diameter is determined to be about 30nm. The double antibody sandwich method was used as the labeled antibody, 2D10 as the capture antibody, and coated on the nitrocellulose membrane. Rabbit anti-mouse IgG antibody was immobilized on C line. The labeling conditions and assembly conditions of the protein were determined. Performance evaluation of test strip. After assembly, the sensitivity of the recombinant protein was determined by detecting the recombinant protein. Only in the detection of natural protein, its sensitivity is reduced. Detection of other proteins secreted by placenta, we can see that the test strip has a good specificity. The stability of the test strip stored at different temperature is good. In this study, the recombinant PSG1 protein and monoclonal antibody with good activity and high purity were obtained, and a test strip with good sensitivity, stability and specificity was prepared for the detection of PSG1 colloidal gold. The next step is to quantify the test strip and further optimize the conditions so that it has a more obvious reaction with the natural PSG1 protein in order to meet the needs of clinical detection.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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