构建人工合成的微小低氧反应元件可调控的AAV载体

Construction of a general AAV vector regulated by minimal and artificial hypoxic-responsive element

目的:人工合成微小HER,将其插入到AAV重组质粒的CMV启动子上游,采用磷酸钙沉淀法包装低氧反应元件调控的AAV重组病毒,为缺血性心脑血管病的基因治疗制备可以控制表达的AAV载体。方法:人工合成具有4次重复串联的低氧诱导因子结合位点(HIF-binding site,HBS)A/GCGTG(4×HBS)的36 bp核酸片段和插入到CMV启动子TATA Box上游的间隔区35 bp的核酸序列,并运用PCR方法扩增。测序正确后,通过常规分子生物学重组方法,将原AAV载体质粒中CMV启动子替换成为含有最小人工合成HRE的CMV启动子,重组构建了低氧反应元件调控的AAV载体。并在该载体CMV启动子下游的多克隆位点插入具有6×His标签的NT4-6His-PR39融合肽序列。磷酸钙沉淀法包装表达NT4-6His-PR39融合肽序列的重组AAV。重组病毒感染人宫颈癌细胞(HeLa),化学性低氧培养,使用免疫组织化学观察低氧条件的诱导表达作用。结果:基因测序结果和酶切结果表明,我们已经把人工合成的HRE,以正确的间隔插入到CMV启动子的上游,成功地构建了HRE调控的AAV重组载体。重组病毒感染的细胞在常氧和低氧诱导表达的结果表明,在启动子上游插入人工合成的微小HRE能够有效调控重组腺相关病毒的表达。结论:使用PCR方法可以将人工合成的HER插入缺乏内切酶识别点的CMV启动子上游,可以制备低氧调控的真核表达载体。人工合成的微小HRE能够有效的调控CMV启动子控制的下游基因表达。该载体的成功制备在缺血性心脑血管病的预防和治疗中具有重要的理论和使用价值。

AIM： To synthesize the minimal and artificial HRE,and to insert it into the anterior extremity of CMV promoter of a AAV plasmid,and then to construct the AAV regulated by hypoxic-responsive element which was introduced into 293 cell by method of Ca3（PO4）2 using three plasmids.Thus obtaining the adenoassociated virus vector regulated by hypoxic-responsive element was possibly used for gene therapy in ischemia angiocardiopathy and cerebrovascular disease.METHODS： Artificially synthesize the 36 bp nucleotide sequences of four connection in series HIF-binding sites A/GCGTG（4×HBS）and a 35 bp nucleotide sequences spacing inserted into anterior extremity of CMV promoter TATA Box,then amplified by PCR.The cDNA fragment was confirmed to be right by DNA sequencing.Molecular biology routine method was used to construct a AAV vector regulated by minimal hypoxic-responsive element after the normal CMV promoter in AAV vector was replaced by the CMV promoter included minimal hypoxic-responsive element.Then,NT4-6His-PR39 fusogenic peptide was inserted into MCS of the plasmid,the recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells,in which we can also investigate the expression of 6×His using immunochemistry in hypoxia environment.RESULTS： Artificial HRE was inserted into anterior extremity of CMV promoter and there was a correct spacing between the HRE and the TATA-box.The DNA sequencing and restriction enzyme digestion results indicated that the AAV regulated by hypoxic-responsive element was successfully constructed.Compared to the control group,the expressions of 6×His was significantly increased in the experimental groups in hypoxia environment,which confirmed that the AAV effectually regulated by the minimal HRE was inserted into anterior extremity of CMV promoter.CONCLUSION： The HRE is inserted into anterior extremity of CMV promoter to lack incision enzyme recognition site by PCR.And eukaryotic expression vector regulated by hypoxic-responsive is constructed.The AAV effectually regulated by the minimal HRE inserted into anterior extremity of CMV promoter.The vector is successfully constructed and it has important theoretical and practical value in the synteresis and therapy of ischemia angiocardiopathy and cerebrovascular disease.