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Alternative TitlePreliminary Study on Preparation of Antibodies against Yeast RNR3 by using Phage Display Technology
Thesis Advisor戴和平
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword核糖核苷酸还原酶 酵母 噬菌体展示技术 单链抗体
Abstract癌症的发生是外界环境因素和自身基因因素共同作用的结果,其中环境因素影响作用超过50%。随着工业化进程加快,环境中导致癌症发生的化学物质的种类和浓度都日益增加, 大多数化学致癌物能导致DNA损伤,而DNA损伤的累积容易引起基因突变,增加患癌风险。由于化学致癌物导致的DNA损伤在癌症发生机制中发挥重要作用,利用响应于DNA损伤的生物标记物检测化学致癌物的存在和暴露剂量是预防癌症的重要策略之一。 核糖核苷酸还原酶(RNRs)存在于已知的一切生物中,参与提供DNA合成底物-脱氧核糖核苷酸,是DNA合成和修复的限速酶。构成酵母RNR的大亚基RNR3具有明显的DNA损伤诱导表达效应,可以发展成为新型化学致癌物DNA损伤效应的生物标记物。抗体是研究蛋白表达水平与诱导信号相互关系的有力工具,也是发展蛋白类分子生物标记物检测方法的基础。为了进一步研究RNR3在细胞内的功能和分布,及其表达水平与DNA损伤信号的响应关系,本研究的主要内容是利用噬菌体展示技术研制识别酵母RNR3的单克隆基因工程抗体。由于RNRs序列的高保守性,易于被识别为自身抗原,所以传统方法难以制备相应单抗,, 而噬菌体展示技术具有可以制备广泛抗原(如毒性抗原、自身抗原和半抗原)的基因工程抗体的优势。为了制备能够广泛识别真核生物RNR大亚基的单克隆基因工程抗体,从而利用抗原-抗体反应检测真核生物RNR大亚基受诱导后表达水平的改变,达到检测DNA损伤物质的目的,该研究通过比对酵母细胞RNR3与其同源性较高的十九个源于真核生物的RNR大亚基蛋白的一级序列,确定了RNR3上一段高保守序列RNR3h,并进行了原核表达;通过构建原核表达RNR3h免疫小鼠的单链抗体噬菌体展示文库,以及非免疫小鼠单链抗体噬菌体展示文库,利用噬菌体展示技术淘选得到识别该原核表达片段的单链抗体。淘选到的单链抗体识别RNR3h原核表达产物空间抗原表位,不识别线性抗原表位。三个特异性识别原核表达RNR3h的单链抗体的基因进行了测序分析,对抗原结合的特异性进行了的研究。研究结果表明,原核表达的抗原,由于对真核蛋白修饰的不完全性,所获得的能识别原核表达蛋白的抗体,并不能保证能识别真核表达的天然蛋白,所以本研究的下一步需要改进的是必须用真核表达的RNR3h来淘选抗体。另外本研究还利用噬菌体展示技术尝试制备分别识别酵母RNR1和RNR3的C端氨基酸序列的单链抗体,构建了两个分别针对不同C端序列的小肽的单链抗体噬菌体展示文库,为今后获取有效的酵母RNR1和RNR3抗体做出了初步尝试,提供了可行的技术路线和改进方向。
Other AbstractCarcinogenesis is related to the external environment factors and genetic factors. Environmental effect weighs over 50% in cancer risk. The chemicals that cause cancer are defined as chemical carcinogen, and the majority of chemical carcinogens can cause DNA damage. The accumulation of DNA damage can cause gene mutations and increase cancer risk. Thus DNA damage plays an important role in carcinogenesis. Using biomarkers in response to DNA-damage for detection of the existence and exposure dose of chemical carcinogens in environment is one of the important strategies in preventing from cancer. Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms, providing all the precursors essential for DNA synthesis. It is the rate-limiting enzyme of both DNA replication and DNA repair. RNR3 is one of big subunit of RNR in Saccharomyces cerevisiae, which has prominent DNA- damage inducible expression effect, and can be developed to be a new biomarker for chemical carcinogen test due to its obvious response to DNA-damage signals. Antibodies are powerful tools in studies on the relationship between the target protein expression level and the induction signals, as well as the base of developing new test method for protein biomarkers. In order to further discover the function and location of yeast RNR3 in vivo and the expression features that induced by DNA damage, we did some preliminary studies in getting monoclonal recombinant antibody with phage display technology. Because of its high conservative structures which may be easily recognized as an autogenous protein, monoclonal antibody is hardly induced against the homologous structure of RNR3 by animal immune system. Whereas genetic manipulated recombinant antibodies to various antigens including self-antigens can be obtained by phage display technology. And the antibodies to RNR3 can help to develop a detection system of chemical carcinogens based on antibody-antigen reactions for biomarker in response to DNA-damage. In this study, a homologous peptide in yeast RNR3 (loc145-loc298) named RNR3h has been selected after comparing the alignment of the RNR3 to 19 homologues peptides in RNRs from other eukaryotes. RNR3h was expressed in E.coli. The purified RNR3h was uesd as antigen for panning against an immuned phage display antibody library and an unimmuned phage display library. ScFvs (single chain Fragment-variable region) that recognise the RNR3h were obtained and further analyzed. The result shows that the scFvs recognize the conformation antigen epitope, but not liner antigen epitope of RNR3h. The genes of three scFvs have been sequenced and their antigen binding specialities were analyzed. The results showed that the antibodies which could recognize procaryotic expressed protein was not guaranted to recognize eukaryotic expressed protein. Thus, the next step to improve the study is using eukaryotic expressed RNR3h to pan against the phage display antibody library. In addition, two phage display libraries against the C-terminal peptide with different amino acid sequence of yeast RNR1 and RNR3 have been constructed; some preliminary investigations have done on finding the antibodies to the two proteins.
Document Type学位论文
Recommended Citation
GB/T 7714
李佳. 利用噬菌体展示技术制备识别酵母RNR3抗体的初步研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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