hVEGF165及hBMP-7双基因共表达腺相关病毒载体的构建及鉴定

CONSTRUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR CO-EXPRESSING hVEGF_(165) AND hBMP-7 GENES

目的通过引入内部核糖体进入位点序列(internal ribosome entry site,IRES),构建带有hVEGF165及hBMP-7双基因的重组腺相关病毒载体(adeno-associated virus,AAV),并对其进行鉴定。方法以AAV腺相关病毒包装系统(helper-free system)为基础,将真核表达质粒pIRES中的IRES片段定向克隆至腺相关病毒骨架质粒pAAV-MCS中,形成含有IRES序列及上、下游多克隆位点的重组骨架质粒pAAV-MCS A-IRES-MCS B。PCR扩增hVEGF165和hBMP-7基因,先后亚克隆入重组腺相关病毒骨架质粒IRES序列上、下游的多克隆位点,获得重组质粒pAAV-hVEGF165-IRES-hBMP-7。将其和包装质粒pAAV-RC、辅助质粒pHelper三质粒共转染AAV-293细胞,包装重组腺相关病毒rAAV-hVEGF165-IRES-hBMP-7,同时包装含有绿色荧光蛋白(green fluorescent protein,GFP)标记的重组病毒rAAV-IRES-GFP作平行对照。荧光显微镜下监测病毒包装效率,收获重组病毒后感染AAV-HT1080细胞测定病毒滴度,并通过病毒基因组外源基因扩增鉴定重组病毒的包装是否成功。结果重组腺相关病毒骨架质粒pAAV-hVEGF165-IRES-hBMP-7经双酶切鉴定正确。荧光显微镜下观察三质粒共转染AAV-293细胞72h后,病毒包装效率达95%～100%,收获的重组病毒具有较高浓度及活性,感染AAV-HT1080效率达90%,荧光计数法测定病毒感染滴度达5.5×1011vp/mL。提取重组病毒基因组成功扩增出外源目的基因hVEGF165及hBMP-7片段,重组病毒包装成功。结论成功构建带有hVEGF165及hBMP-7双基因的重组腺相关病毒rAAV-hVEGF165-IRES-hBMP-7,收获的病毒具有较高滴度,为今后利用腺相关病毒载体进行hVEGF165及hBMP-7双基因共表达影响骨修复的体外及体内研究提供实验基础。

Objective To construct the recombinant adeno-associated virus （rAAV） vector co-expressing hVEGF165 and hBMP-7 depending on internal ribosome entry site （IRES） sequence, to measure the virus titer and to verify the correct recombination. Methods The AAV helper-free system was used to generate the rAAV co-expressing hVEGF165 and hBMP-7 genes. The IRES sequence from the bicistronic eukaryotic expression plasmid plRES was cut down and subcloned into the ITR/MCS containing vector pAAV-MCS to get pAAV-MCS A-IRES-MCS B, in which upstream MCS A and downstream MCS B was constructed. The hVEGF165 and hBMP-7 genes were amplified by PCR and inserted into MCS A and MCS B respectively. The recombinant expression plasmid pAAV-hVEGF165-IRES-hBMP-7 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recombinant AAV. The green fluorescent protein （GFP） labeled rAAV- IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-GFP. The efficiency of rAAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured through infecting AAV-HT1080 cells, and the recombinant rAAV-hVEGF165-IRES-hBMP-7 was verified by PCR of the exogenous interest genes of hVEGF165 and hBMP-7. Results The recombinant plasmid pAAV- hVEGF165-IRES-hBMP-7 was verified by double digestion. Using the AAV helper-free system, GFP expression could be observed under fluorescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 95% -100% . The rAAV has a high purity and high titer of 5.5 × 10611 vp/mL, and AAV-HT1080 cell could be infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP-7 and hVEGF165 genes. Conclusion Re combinant rAAV-hVEGF165-IRES-hBMP-7 was successfully constructed with a high virus titer, which may offer the basement of in vitro and in vivo experiments of hVEGFI65 and hBMP-7 co-expressing for gene therapy of bone regeneration.