文档介绍：
利用 DNA Shuffling 技术进化靶向卵巢癌细
胞的腺相关病毒#
张悦1,2,潘素晶1,2,钱其军1,2**
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(1. 浙江理工大学生命科学学院新元医学与生物技术研究所,杭州 310018;
2. 第二军医大学东方肝胆医院病毒基因治疗实验室,上海 200438)
摘要:在过去的几年里,重组腺相关病毒(rAAV)作为基因治疗载体,在转导肝脏,心脏
以及视网膜等方面取得了可观的成果。然而,AAV 病毒的宿主广泛,这使其对组织和细胞的
靶向能力较差,因而通过基因工程的手段改造 AAV 病毒,来提高它对目的细胞的靶向能力和
感染效率,是现阶段的研究重点。近年来,科学家已通过一系列的手段改造 AAV 衣壳蛋白,
以此改变病毒的靶向性。DNA shuffling 是一种体外定向进化技术,其通过对来源不同但功
能相近的同源基因的核酸序列进行改造,创造出新基因,赋予其表达产物新功能。本实验利
用 DNA shuffling 技术改造了 8 种 AAV 的衣壳基因,建立了嵌合型质粒文库,通过转染 HEK293
细胞,获得了嵌合型衣壳蛋白包被的 AAV,构建出高多样性的病毒文库。经过 5 轮体外筛选,
成功获得了能够有效感染卵巢癌细胞 SK-OV-3f 的新型 qAAV 病毒。序列分析表明其为 AAV1,
2,3 和 8 型的嵌合体。随后以 3 种其他组织细胞为对照,进行靶向性实验,结果证明了筛
选得到的嵌合型病毒对 SK-OV-3f 细胞具备一定的靶向性。
关键词:腺相关病毒;DNA shuffling 技术;体外筛选;卵巢癌
中图分类号:Q782
An ovarian cancer tropic adeno-associated virus evolved by
DNA Shuffling
ZHANG Yue1,2, PAN Sujing1,2, QIAN Qijun1,2
(1. Xinyuan Institute of Medicine and Biotechnology,School of Life Sciences, Zhejiang Sci-Tech
University, Hangzhou 310018, Zhejiang, China;
2. Laboratory of Virus and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, Second
Military Medical University, Shanghai 200438, China)
Abstract: In the past decades, rAAV has been used broadly as a vector of gene therapy to
transduce liver, muscle, retina, etc. with great achievements. However, the wide host range of
AAV results in a problem that AAV lacks the specificity to tissue or cell. Thus, it is crucial to
improve the targeting and infection of AAV to cell through AAV transformation by ic
engineering. In recent years, scientists have transformed the capsid protein to change the tropism
of AAV by a series of methods. DNA shuffling is a technic of directed evolution in vitro. Through
modifying homologous genes with similar function and different sources, this method can create
new gene which expresses the protein with new function. This experiment transformed the capsid
gene of eight AAVs by DNA shuffling, and constructed a mosaic plasmid library. Transfecting
HEK239