Studies of lentiviral system for gene transferring and its expansile application

慢病毒载体因为具有能感染分裂和未分裂的宿主细胞，并且带给宿主细胞轻微的免疫原性的优点，而在转基因改造、基因功能分析和基因治疗中得到了广泛的应用。但是，慢病毒载体的整合特性，尤其是它整合到基因组的转录活性区域或邻近活性区域的特性，使基因功能分析的结果产生未知干扰，并给实际应用带来风险。慢病毒的整合可能会引起突变，或者改变非目的基因的表达从而对目的基因的功能研究产生影响，而且病毒序列整合到宿主细胞基因组中可能会在转基因操作中带来长期和有害的影响。我们的研究旨在利用慢病毒的优良特性的同时，通过其他的改进措施来解决慢病毒整合带来的消极影响，拓展慢病毒的应用性。 为了避免插入突变的风险，我们对慢病毒包...Lentiviral vectors have been extensively implicated in transgenic modification, gene function analysis and gene therapy because they are able to infect a wide range of dividing and non-dividing host cells with weak immunogenicity. However, their property of integration, preferentially to integrate into or close to transcriptionally active genome regions, gives rise to big puzzle for gene function ...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：2162007015382

Prokaryotic expression,purification of chicken Rad51 protein and generation of its antiserum

目的:rAd51蛋白在双链断裂的dnA修复的同源重组中发挥重要作用。因而其过量表达在细胞内的基因操作中被用于在提高基因同源重组的效率。当前在鸡中还未有针对该蛋白的特异性抗体。为此我们对鸡的rAd51进行了体外表达与纯化,并制备了CrAd51抗体,为该蛋白质在鸡细胞中的功能研究及探讨其用于鸡细胞中提高同源重组的研究提供有效工具。方法:对鸡rAd51基因进行全长克隆并构建原核表达载体PET-28A(+),用IPTg诱导该蛋白在rOSSETA菌株中表达,纯化后注射bAlb/C小鼠获得抗体。结果:成功制备鸡rAd51特异多克隆抗体,ElISA显示该抗体血清效价达51 200,该抗体血清可以用于WESTErn等的蛋白检测技术。结论:针对鸡基因产物制备抗体的技术方法有效可行;首次成功获得鸡rAd51蛋白及多克隆抗体,为与该蛋白相关的研究提供了有效工具。Objective:RAD51 protein plays a major role in homologous recombination of DNA double strand break repair.Overexpression of this protein has thus been used to enhance the homologous recombination efficiency in gene manipulations in cells.Currently,there is not yet an antibody available for this chicken protein.We therefore carried out in vitro expression,purification of the chicken Rad51,and tried to prepare the antibody.Methods:Full length of chicken Rad51 coding sequence was cloned and inserted into pET-28a(+) to generate a prokaryotic expression constructs;the expression was induced with IPTG in a Rosseta strain.After the purification,the gene product was procedurally injected into BALB/c mouse to generate antibody.Results:ELISA test showed that the titer of antiserum reached 51200;The antiserum had a good performance in Western blot for both sensitivity and specifity.Conclusion:The protocol adapted in this study works well in generating antiserum against chicken gene product;We have for the first time prepared an antiserum against chicken Rad51 providing a useful tool for studies on related to the chicken Rad51.科技部重大基础研究计划资助(2009CB941601