本文選題：塵肺 + 噬菌體抗體庫��； 參考：《鄭州大學(xué)》2011年碩士論文

【摘要】：研究背景 塵肺病目前仍是中國職業(yè)病中發(fā)病率最高、死亡危險(xiǎn)性較大、對勞動者危害最嚴(yán)重的一類,迄今尚無將其消除或逆轉(zhuǎn)的根治方法,只能根據(jù)病情進(jìn)行綜合醫(yī)治以減輕癥狀、延緩病情進(jìn)展,因此,積極預(yù)防并盡早診斷對于增進(jìn)病人生存質(zhì)量及延長壽命尤為重要。迄今為止,國內(nèi)外已經(jīng)對塵肺病進(jìn)行了大量的相關(guān)研究,但塵肺病的形成機(jī)制目前還不十分清楚,不僅塵肺病所致肺組織纖維化尚無消除或逆轉(zhuǎn)的根治方法,也缺乏接塵人群健康監(jiān)護(hù)、早期預(yù)警及塵肺病早期發(fā)現(xiàn)的有效生物監(jiān)測指標(biāo),診斷仍然依靠達(dá)到一定病理程度后的x線表現(xiàn)。進(jìn)一步探索塵肺病的發(fā)生、發(fā)展和演進(jìn)過程,篩選和鑒定出能夠反映粉塵接觸、塵肺病進(jìn)程的生物標(biāo)志,對解釋塵肺病發(fā)病分子機(jī)制、進(jìn)行塵肺發(fā)病的早期預(yù)警和診斷以及研究阻止纖維化進(jìn)展的干預(yù)靶點(diǎn),都具有積極的意義和作用。 目的 利用噬菌體抗體庫技術(shù),構(gòu)建人源塵肺病ScFv噬菌體單鏈抗體庫,為塵肺病生物標(biāo)記篩選打下基礎(chǔ)。 方法 首先分離塵肺病人外周血淋巴細(xì)胞,提取總RNA,反轉(zhuǎn)錄合成第一鏈cDNA,采用兼并引物利用半巢式PCR擴(kuò)增抗體重鏈(VH)和輕鏈可變區(qū)(VL)基因。使用帶有粘端的Linker將VH和VL連接成單鏈抗體(ScFv)基因。 將ScFv和噬菌粒載體pCANTAB-5E進(jìn)行雙酶切后,將ScFv與pCANTAB-5E進(jìn)行拼接,轉(zhuǎn)化至感受態(tài)大腸桿菌TGl,經(jīng)輔助噬菌體超感染, 構(gòu)建了全人源塵肺噬菌體單鏈抗體庫,對抗體庫的庫容量與多樣性進(jìn)行檢測。 結(jié)果 結(jié)果顯示：瓊脂糖電泳從塵肺病人外周血中提取的總RNA,可見明顯的28S和18S 2條帶,提示總RNA無降解且完整性較好；VH片段大小為370bp左右,VL片段為320bp左右,組裝后ScFv基因片段(VH-Linke-VL)長度為750bp左右。 涂板后隔夜培養(yǎng),初步估算抗體庫庫容量為2.4×107的噬菌體單鏈抗體庫。隨機(jī)挑取5個單克隆,質(zhì)粒雙酶切后顯示陽性插入率100%(5/5)。 結(jié)論：成功構(gòu)建了庫容為2.4×107的全人源塵肺病噬菌體單鏈抗體庫,抗體庫多樣性良好。
[Abstract]:Research background At present, pneumoconiosis is still the highest incidence of occupational disease in China, which has the highest risk of death and the most serious harm to workers. So far, there is no radical cure to eliminate or reverse pneumoconiosis, so it can only be treated comprehensively according to the condition to alleviate the symptoms. Therefore, active prevention and early diagnosis are of great importance in improving the patient's quality of life and prolonging life span. Up to now, there have been a lot of research on pneumoconiosis, but the mechanism of pneumoconiosis is not clear, not only there is no radical cure method to eliminate or reverse pulmonary fibrosis caused by pneumoconiosis. There is also a lack of effective biological monitoring indicators such as health monitoring early warning and early detection of pneumoconiosis. The diagnosis of pneumoconiosis still depends on the X-ray manifestations after reaching a certain pathological level. To further explore the occurrence, development and evolution of pneumoconiosis, to screen and identify the biomarkers that can reflect the process of dust exposure and pneumoconiosis, and to explain the molecular mechanism of pneumoconiosis. The early warning and diagnosis of pneumoconiosis and the study of intervention targets to prevent the progression of fibrosis have positive significance and role. Purpose The ScFv phage single chain antibody library of human pneumoconiosis was constructed by using phage antibody library technology, which laid the foundation for screening biological markers of pneumoconiosis. Method First, peripheral blood lymphocytes of pneumoconiosis patients were isolated, total RNAs were extracted, and the first strand cDNAs were synthesized by reverse transcription. The antibody heavy chain (PCR) and light chain variable region (PCR) genes were amplified by degenerate primer using semi-nested PCR. VH and VL were ligated into scFv gene using Linker with sticky terminal. ScFv and pCANTAB-5E were digested by double enzyme, then ScFv and pCANTAB-5E were spliced together, and then transformed into TGls of Escherichia coli, which was infected by bacteriophage. A phage single chain antibody library was constructed to detect the capacity and diversity of the antibody library. Result The results showed that the total RNAs extracted from the peripheral blood of pneumoconiosis patients by agarose electrophoresis showed obvious 28s and 18s bands, indicating that the total RNA was not degraded and the complete VH fragment was about 320bp. The length of ScFv gene fragment VH-Linke-VLL is about 750bp. The phage scFv library with a capacity of 2.4 脳 107 was preliminarily estimated after overnight culture. Five monoclonal clones were randomly selected and the positive insertion rate was 100% after double enzyme digestion. Conclusion: the phage single-chain antibody library with a capacity of 2.4 脳 107 has been successfully constructed, and the diversity of antibody library is good.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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