本文選題：妊娠特異性β1糖蛋白 + 單克隆抗體��； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：妊娠特異性β1糖蛋白（Pregnancy Specific β1Glycoprotein, PSG1）是妊娠期間由胎盤滋養(yǎng)層細胞合成的一種蛋白質(zhì)，合成后外分泌到母體外周血液循環(huán)中。在母體血清中PSG1低于正常值的情況下，，常常造成一些不良后果，如胎兒發(fā)育不良及流產(chǎn)等[1]。同樣病理狀態(tài)下，PSG1也被發(fā)現(xiàn)存在于許多腫瘤組織中。作為一種妊娠的特異性糖蛋白，PSG1具有監(jiān)測胎盤功能、檢測胎兒生長受限、檢測腫瘤細胞、參與免疫調(diào)節(jié)、以及維持妊娠期間母體的免疫耐受環(huán)境等功能[2]。一般認(rèn)為,在早孕的診斷、胎盤功能的監(jiān)測、以及胎兒預(yù)后的判定等一些應(yīng)用上，PSG1蛋白含量的高低可以作為結(jié)果判斷的一個依據(jù)。因此對PSG1的研究也越來越受到重視。先前工作中，實驗室已經(jīng)構(gòu)建了能穩(wěn)定表達PSG1單抗的雜交瘤細胞株、重組蛋白的原核表達系統(tǒng)。在此基礎(chǔ)上，從中選擇兩種抗原表面結(jié)合位點之間影響小的細胞株。采用雙抗體夾心法的原理，以建立一種靈敏特異、快速簡便的PSG1檢測方法。 1. PSG1重組蛋白的表達和純化 復(fù)蘇具有原核表達質(zhì)粒的菌株DE3，并擴大培養(yǎng)，制備包涵體蛋白。復(fù)性、純化后，用于后續(xù)的檢測。純化后蛋白的濃度為0.6mg/ml。 2. PSG1單抗細胞株的篩選。 復(fù)蘇3株單抗細胞株，并對其穩(wěn)定性進行檢測。用疊加法ELISA實驗對實驗室制備的3株單抗細胞株進行篩選，挑選出抗原識別表位影響小的兩株單抗9G6和2D10。 3.單克隆抗體的制備以及純化。 取健康小鼠，注射雜交瘤細胞在腹腔中。取腹水來制備單抗，離心后取上清，除去雜質(zhì)以及表面脂質(zhì)。然后用G蛋白進一步純化抗體。SDS-PAGE電泳及WB檢測純化效果。測定其蛋白濃度2D10和9G6分別為1.69mg/ml，1.37mg/ml。ELISA檢測抗體的效價大于1:2.48×106。 4.制備膠體金，確定標(biāo)記條件及組裝條件。制備膠體金。通過檢測其吸收光波長，判斷其直徑在30nm左右。采用雙抗體夾心法，9G6作為標(biāo)記抗體，2D10作為捕獲抗體，包被于硝酸纖維素膜上。兔抗小鼠IgG抗體固定在C線上。并確定了蛋白的標(biāo)記條件以及組裝條件。 5.試紙條的性能評價。 組裝后，通過對重組蛋白的檢測，判斷其靈敏度較好。只是在檢測天然蛋白時，其靈敏度的降低。檢測與胎盤分泌的其他蛋白，可以看出試紙條有較好的特異性。檢測不同溫度下保存的試紙條，其穩(wěn)定性良好。 本研究獲得了活性較好，純度較高的重組PSG1蛋白以及單克隆抗體，制備了具有良好靈敏度，穩(wěn)定性以及特異性的PSG1膠體金檢測試紙條。下一步需要做的是將檢測試紙條定量化，進一步優(yōu)化條件，使之與天然PSG1蛋白有更加明顯的反應(yīng)，以滿足臨床檢測的需要。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.6

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