人源尘肺病噬菌体单链抗体库的构建和初步鉴定

发布时间：2018-05-17 07:39

本文选题：尘肺 + 噬菌体抗体库　；参考：《郑州大学》2011年硕士论文

【摘要】：研究背景尘肺病目前仍是中国职业病中发病率最高、死亡危险性较大、对劳动者危害最严重的一类,迄今尚无将其消除或逆转的根治方法,只能根据病情进行综合医治以减轻症状、延缓病情进展,因此,积极预防并尽早诊断对于增进病人生存质量及延长寿命尤为重要。迄今为止,国内外已经对尘肺病进行了大量的相关研究,但尘肺病的形成机制目前还不十分清楚,不仅尘肺病所致肺组织纤维化尚无消除或逆转的根治方法,也缺乏接尘人群健康监护、早期预警及尘肺病早期发现的有效生物监测指标,诊断仍然依靠达到一定病理程度后的x线表现。进一步探索尘肺病的发生、发展和演进过程,筛选和鉴定出能够反映粉尘接触、尘肺病进程的生物标志,对解释尘肺病发病分子机制、进行尘肺发病的早期预警和诊断以及研究阻止纤维化进展的干预靶点,都具有积极的意义和作用。目的利用噬菌体抗体库技术,构建人源尘肺病ScFv噬菌体单链抗体库,为尘肺病生物标记筛选打下基础。方法首先分离尘肺病人外周血淋巴细胞,提取总RNA,反转录合成第一链cDNA,采用兼并引物利用半巢式PCR扩增抗体重链(VH)和轻链可变区(VL)基因。使用带有粘端的Linker将VH和VL连接成单链抗体(ScFv)基因。将ScFv和噬菌粒载体pCANTAB-5E进行双酶切后,将ScFv与pCANTAB-5E进行拼接,转化至感受态大肠杆菌TGl,经辅助噬菌体超感染, 构建了全人源尘肺噬菌体单链抗体库,对抗体库的库容量与多样性进行检测。结果结果显示：琼脂糖电泳从尘肺病人外周血中提取的总RNA,可见明显的28S和18S 2条带,提示总RNA无降解且完整性较好；VH片段大小为370bp左右,VL片段为320bp左右,组装后ScFv基因片段(VH-Linke-VL)长度为750bp左右。涂板后隔夜培养,初步估算抗体库库容量为2.4×107的噬菌体单链抗体库。随机挑取5个单克隆,质粒双酶切后显示阳性插入率100%(5/5)。结论：成功构建了库容为2.4×107的全人源尘肺病噬菌体单链抗体库,抗体库多样性良好。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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