本文選題：噬菌體抗體庫(kù) + 精氨酸加壓素��； 參考：《天津醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目前小分子激素的檢測(cè)相對(duì)困難,這極大地影響了對(duì)復(fù)雜內(nèi)分泌現(xiàn)象的認(rèn)識(shí)和內(nèi)分泌疾病的診斷。很多年來,我們對(duì)相關(guān)內(nèi)分泌疾病只能通過間接推測(cè)作出診斷。如下丘腦、垂體疾病中尿崩癥的診斷僅能以尿量、尿比重、尿滲透壓等間接指標(biāo)反映體內(nèi)精氨酸加壓素的水平,因此建立體內(nèi)小分子激素的快速檢測(cè)方法對(duì)于臨床上疾病的診治具有重要價(jià)值。由于這些激素的分子量小,半衰期短,傳統(tǒng)制備抗體的方法如雜交瘤技術(shù)和機(jī)體免疫的方法制備抗體得到的抗體少,且工作繁瑣,缺乏穩(wěn)定性不宜大規(guī)模生產(chǎn),因此尋找一種快速、簡(jiǎn)單制備精氨酸加壓素抗體的方法是開展進(jìn)一步研究的關(guān)鍵環(huán)節(jié)。 噬菌體抗體庫(kù)技術(shù)是在基因工程技術(shù)基礎(chǔ)上發(fā)展起來的一種模擬自然免疫選擇系統(tǒng)制備抗體的新技術(shù)。它將抗體的基因型和表現(xiàn)型統(tǒng)一于一體,使其識(shí)別抗原的能力和噬菌體的擴(kuò)增能力偶聯(lián)起來,具有強(qiáng)大的篩選能力。它不需人工免疫動(dòng)物,繞過雜交瘤技術(shù),用基因工程技術(shù)制備人源性抗體,較好地解決了人雜交瘤技術(shù)低效及人體排異反應(yīng)的難題,在制備難以獲得的抗體方面具有很大的潛力。近些年,噬菌體抗體庫(kù)技術(shù)已廣泛應(yīng)用于生命科學(xué)的各個(gè)領(lǐng)域,但利用該技術(shù)制備小分子激素抗體的報(bào)道少見。本研究以精氨酸加壓素為模型分子,探索利用噬菌體抗體庫(kù)技術(shù)從構(gòu)建的人源性天然噬菌體Fab抗體庫(kù)中直接篩選精氨酸加壓素特異性Fab抗體,并進(jìn)行可溶性表達(dá)及免疫活性鑒定,建立小分子激素抗體的制備方法,為下一步的臨床研究奠定了基礎(chǔ)。 方法：以純化的精氨酸加壓素為靶抗原,梯度稀釋濃度包被到酶標(biāo)板上,應(yīng)用噬菌體抗體庫(kù)技術(shù),從庫(kù)容為2.4×108的人源噬菌體抗體庫(kù)中篩選抗精氨酸加壓素Fab抗體。采用固相篩選法,經(jīng)過5輪“吸附-洗脫-擴(kuò)增”富集篩選。Phage-ELISA鑒定精氨酸加壓素特異性噬菌體抗體,挑選結(jié)合活性最強(qiáng)的陽(yáng)性克隆,制備噬菌體上清,感染大腸桿菌HB2151,經(jīng)IPTG誘導(dǎo)高效表達(dá),表達(dá)的可溶性抗體經(jīng)SDS-PAGE、Western Blot檢測(cè)鑒定抗體的表達(dá)情況,ELISA鑒定其抗原結(jié)合活性和特異性。 結(jié)果：經(jīng)過5輪富集篩選,最終篩選出6株具有較強(qiáng)特異結(jié)合活性的抗精氨酸加壓素抗體。其中,陽(yáng)性克隆C4展示了最強(qiáng)的結(jié)合活性。挑選陽(yáng)性克隆C4在大腸桿菌HB2151中誘導(dǎo)可溶性表達(dá),表達(dá)的抗體片段經(jīng)SDS-PAGE電泳和Western Blot檢測(cè),在相對(duì)分子質(zhì)量約為50kD附近有特異條帶的出現(xiàn),同預(yù)計(jì)的目的蛋白分子量大小一致,ELISA證實(shí)該抗體片段具有較好的抗原特異性和結(jié)合活性。 結(jié)論：本研究從自建的人源性Fab噬菌體抗體庫(kù)中篩選出抗精氨酸加壓素抗體,并對(duì)抗體的有效性進(jìn)行了初步鑒定,構(gòu)建了精氨酸加壓素天然抗體制備技術(shù)的平臺(tái),為將來更多小分子激素快速檢測(cè)方法的建立奠定了良好的基礎(chǔ)。
[Abstract]:At present, the detection of small molecular hormones is relatively difficult, which greatly affects the understanding of complex endocrine phenomena and the diagnosis of endocrine diseases. For many years, we diagnosed endocrine diseases only through indirect speculation. For example, the diagnosis of diabetes insipidus in hypothalamus and pituitary diseases can only reflect the level of arginine vasopressin by indirect indexes such as urine volume, specific gravity and osmotic pressure of urine. Therefore, the establishment of a rapid detection of small molecular hormones in vivo is of great value in the diagnosis and treatment of diseases. Because of the small molecular weight and short half-life of these hormones, the traditional methods of preparing antibodies, such as hybridoma and immune methods, have less antibodies, and the work is tedious, so the lack of stability is not suitable for mass production. Therefore, to find a rapid and simple method to prepare arginine vasopressin antibody is the key to further research. Phage antibody library (Phage antibody library) is a new technique which is developed on the basis of genetic engineering and simulates natural immune selection system to prepare antibodies. It unifies the genotype and phenotype of antibody, and makes the ability of antigen recognition and phage amplification coupling together, and has a strong screening ability. It does not need artificial immunity to animals, bypasses hybridoma technology, and uses genetic engineering technology to prepare human antibody, which solves the problem of low efficiency of human hybridoma technology and rejection of human body. There is great potential in the preparation of difficult-to-obtain antibodies. In recent years, phage antibody library technology has been widely used in various fields of life science, but the preparation of small molecular hormone antibodies by using this technique is rare. In this study, arginine vasopressin was used as model molecule to screen arginine vasopressin specific Fab antibody directly from human natural phage Fab antibody library by phage antibody library technology. The soluble expression and immunological activity were identified, and the preparation method of small molecular hormone antibody was established, which laid a foundation for clinical research in the next step. Methods: the purified arginine vasopressin was used as the target antigen and the gradient dilution concentration was coated on the enzyme marker. The anti-arginine vasopressin Fab antibody was screened from the library containing 2.4 脳 10 ~ 8 human phage antibody library by phage antibody library technique. The specific phage antibody of arginine vasopressin was identified by 5 rounds of "adsorption-eluation-amplification" enrichment screening. The phage supernatant was prepared by selecting the positive clones with the strongest binding activity. E. coli HB2151 was infected and highly expressed by IPTG. The expressed soluble antibody was detected by SDS-PAGEG Western Blot and its antigen-binding activity and specificity were identified by Elisa. Results: after 5 rounds of enrichment screening, 6 strains of anti-arginine vasopressin antibodies with strong specific binding activity were screened. Among them, the positive clone C4 showed the strongest binding activity. The positive clone C4 was selected to induce soluble expression in Escherichia coli HB2151. The expressed antibody fragment was detected by SDS-PAGE electrophoresis and Western Blot, and there were specific bands near the relative molecular weight of 50kD. In accordance with the predicted molecular weight of the target protein, Elisa confirmed that the antibody fragment had good antigen-specificity and binding activity. Conclusion: the anti-arginine vasopressin antibody was screened from the human Fab phage antibody library, and the effectiveness of the antibody was preliminarily identified, and a platform for the preparation of arginine vasopressin natural antibody was established. It has laid a good foundation for the establishment of more rapid detection methods of small molecular hormones in the future.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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