血液细胞靶向性腺病毒载体的构建及功能检测

发布时间：2018-05-22 08:01

本文选题：血液细胞 + 靶向　；参考：《安徽医科大学》2006年硕士论文

【摘要】：目前基因转运方法很多，其中包括物理方法、化学方法、融合法、基因枪等。上述基因转移方法各有优点，但共同问题是基因转移效率低，较难获得稳定表达的细胞。腺病毒载体由于具有宿主范围广、能制备高滴度病毒、感染效率高、高表达目的基因、能感染增殖及静止两类细胞、不与宿主细胞基因组DNA结合、容许插入大片段外源基因等特点而受到人们的青睐。多数用于基因转移的腺病毒载体都来自于5型或2型腺病毒(Ad5和Ad2)，，但很多血液系统来源的细胞对腺病毒感染具有天然的抵抗性，因此限制了该载体在造血研究领域的应用。腺病毒纤维蛋白在腺病毒黏附到靶细胞的过程中起关键作用，不同腺病毒亚型可能会具有不同的组织或细胞趋向性，因为它们的纤维蛋白会识别不同的细胞表面受体。血液细胞表面具有B组腺病毒Ad11p的高效结合受体，可以通过对5型腺病毒载体进行纤维改造，使其具有B组腺病毒的趋向性，从而增强对血液细胞的基因转移效率。本研究以AdEasy系统为基础，先用递归PCR的方法人工合成人B组11p型腺病毒的部分纤维(fiber)基因，测序正确后，经过多次的酶切连接操作，将其替换AdEasy骨架质粒中的人5型腺病毒的fiber基因，得到新的腺病毒骨架质粒命名为AdEasy-1／F11p，应用带有绿色荧光蛋白(green fluorescence protein,GFP)报告基因的穿梭质粒pShuttle-GFP与AdEasy-1／F11p腺病毒DNA在BJ5183细菌内进行同源重组得到重组腺病毒质粒，切取重组腺病毒基因组DNA，脂质体转染法将其转运到293包装细胞内获得重组腺病毒，命名为Ad5F11p-GFP。同时以GFP监测腺病毒载体包装、扩增及滴度测定。为了检测改建后的重组腺病毒感染血液细胞的能力，以Ad5-GFP作对照，应用纯化后的重组腺病毒感染白血病细胞系、原代白血病细胞以及脐带血来源的CD34~+造血干细胞，流式细胞仪检测GFP的表达。结果显示这种改建后的重组腺病毒能靶向结合血液细胞，其转染血液细胞的能力明显提高，显示出很好的应用前景。
[Abstract]:There are many methods of gene transport, including physical method, chemical method, fusion method, gene gun and so on. These methods have their own advantages, but the common problem is that the efficiency of gene transfer is low and it is difficult to obtain stable expression cells. Because of its wide host range, adenovirus vector can prepare high titer virus, high infection efficiency, high expression of target gene, can infect both proliferative and stationary cells, and does not bind to host cell genomic DNA. Allowing the insertion of large fragments of foreign genes and other characteristics are favored by people. Most of the adenovirus vectors used for gene transfer come from adenovirus type 5 or type 2 adenovirus, but many cells from blood system have natural resistance to adenovirus infection, so the application of adenovirus vector in hematopoietic research is limited. Adenovirus fibrin plays a key role in the process of adenovirus adhesion to target cells. Different adenovirus subtypes may have different tissue or cell tendency because their fibrin recognizes different cell surface receptors. The surface of blood cells has a highly efficient binding receptor of group B adenovirus Ad11p, which can be modified with the fiber of adenovirus type 5 to make it have the tendency of group B adenovirus, thus enhancing the efficiency of gene transfer to blood cells. In this study, based on AdEasy system, the human B group 11p adenovirus partial fiber-fibergene was synthesized by recursive PCR method. After correct sequencing, the gene was digested and ligated several times. To replace the fiber gene of human adenovirus type 5 in the AdEasy skeleton plasmid. A new adenovirus skeleton plasmid named AdEasy-1 / F11p was obtained. The recombinant adenovirus plasmid pShuttle-GFP and AdEasy-1/F11p adenovirus DNA were homologous recombined in BJ5183 bacteria by using the shuttle plasmid pShuttle-GFP with the green fluorescent protein fluorescence protein (GFP) reporter gene. The recombinant adenovirus DNA was digested and transferred into 293 packaging cells by liposome transfection to obtain the recombinant adenovirus named Ad5F11p-GFP. At the same time, GFP monitoring adenovirus vector packaging, amplification and titer determination. In order to test the ability of recombinant adenovirus to infect blood cells, the purified recombinant adenovirus was used to infect leukemia cell lines, primary leukemia cells and CD34 ~ hematopoietic stem cells derived from umbilical cord blood, using Ad5-GFP as control. The expression of GFP was detected by flow cytometry. The results showed that the recombinant adenovirus could target blood cells, and its ability to transfect blood cells was significantly improved, which showed a good prospect of application.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

【共引文献】