通用型腺病毒伴随病毒载体的构建

The construction of a general adeno-associated virus vector

目的构建含腺病毒伴随病毒（ＡＡＶ）基因组两端的反向重复序列（ＩＴＲｓ）和表达必须元件如启动子、多克隆位点和ＰｏｌｙＡ信号的通用型载体质粒ｐＡＣＲ－Ｎｅｏ，并获得重组ＡＡＶ（ｒＶＶ／ＡＣＲ－Ｎｅｏ）。方法通过ＤＮＡ重组技术，将ＳＶ４０ＰｏｌｙＡ、Ｎｅｏ基因、ＣＭＶ－ＩＥ启动子和多克隆位点组成表达盒子，取代含ＡＡＶ全基因组质粒ｐＳＳＶ９中ＡＡＶ结构基因部分，构建成质粒ｐＡＣＲ－Ｎｅｏ。用ｐＡＣＲ－Ｎｅｏ转染５型腺病毒（Ａｄ５）感染的重组ＡＡＶ包装细胞系ＡＥ１２０１，能获得重组病毒ｒＡＡＶ／ＡＣＲ－Ｎｅｏ。提取ｒＡＡＶ／ＡＣＲ－Ｎｅｏ感染细胞的染色体，用Ｓｏｕｔｈｅｍ杂交分析重组病毒基因组在感染细胞中的存在情况。结果质粒ｐＡＣＲ－Ｎｅｏ转染包装细胞系后所得ｒＡＡＶ／ＡＣＲ－Ｎｅｏ滴度为４．２×１０５ＣＦＵ／ｍｌ，并且ｒＡＡＶ／ＡＣＲ－Ｎｅｏ能在转导细胞内实现其基因组与细胞染色体的整合。结论成功地构建了通用型ＡＡＶ载体，为今后的ＡＡＶ载体研究。

The general adeno-associated virus(AAV)vector pACR-Neo was constructed by substituting AAV's construct gene with cassette that was composed of CMV-IE promoter,multiple cloning sites and SV40-polyA.After transfecting pACR-Neo into recombinant AAV's packaging cell line AE1201,which was infected by Adenovirus 5 before transfection,we got rAAV/ACR-Neo at the titer of 4.2×105 CFU/ml.Using rAAV/ACR-Neo infected host cell A549,the recombinant AAV could transfer reporter gene Neo into host cell and mediate the integration of viral genome into host cell's choromosome DNA.By extracting choromosome DNA of host cell A549 that had been infected by rAAV/ACR-Neo,and digestion respectively with restriction endonuclease PvuII and HindIII,each of which has only one cutting site within rAAV/ACR-Neo's genome.We did Southern blot to check the hybridization of endonuclease digested choromosome to digoxin labelled Neo gene probe.After analyzing the experimental results,we found that rAAV/ACR-Neo's genome had integrate into host cell's choromosome compared to wild type AAV's site-specific integration.Through this work,we constructed a general AAV vector successfully.It lays foundation for research on AAV vector and application on gene therapy.