过表达HGF基因AAV9型腺相关病毒载体的构建与鉴定

Construction and Identification of AVV9 with Overexpression of HGF Gene in Mice

目的构建稳定表达肝细胞生长因子(hepatocyte growth factor,HGF)基因重组腺相关病毒过表达质粒载体,制备并纯化HGF过表达的高滴度重组腺相关病毒。方法合成HGF基因并利用PCR扩增,退火形成双链,与pAAV-ITR-CMV质粒经XhoⅠ单酶切连接构建质粒pAAV-ITR-CMV-AAV9-HGF,转化至大肠杆菌DH5α,提质粒后经酶切和测序鉴定证实重组质粒克隆正确,将重组质粒转染至293FT细胞中包装为腺相关病毒。小鼠注射病毒后通过Western blot法评价HGF的蛋白表达水平。结果扩增产物包含HGF CDS全序列的2186 bp,酶切结果与测序结果说明PCR扩增HGF目的片段成功;实时荧光定量PCR检测结果显示,纯化后所得的过表达HGF的腺相关病毒滴度为5.22×10^(12) vg·mL^(-1);HGF过表达组HGF蛋白相对表达含量较生理盐水组与阴性对照组均升高(P均<0.01)。结论本研究成功构建了HGF腺相关病毒载体且病毒滴度达到普通动物注射要求,证明腺相关病毒载体构建成功。

Objective This study was aimed to construct the stable expression of Hepatocyte Growth Factor(HGF)gene recombinant adeno-associated virus overexpression plasmid vector,and to prepare and purify HGF-overexpression high-titer adeno-associated virus.Methods First,the HGF gene was synthesized and amplified by PCR.The double strands were linked with the pAAV-ITR-CMV plasmid by XhoⅠto construct the plasmid pAAV-ITR-CMV-AAV9-HGF,which was transformed into escherichia coli DH5α.The recombinant plasmid was identified correctly by enzyme digestion and sequencing.The recombinant plasmid was transfected into 293FT cells and packaged as adeno-associated virus.The protein expression level of HGF was evaluated by Western blot after virus injection in mice.Results The amplification product contained HGF CDS 2186 bp and the results of restriction endonuclease digestion and sequencing showed that the target fragment of HGF was successfully amplified by PCR.The results of real-time fluorescence quantitative PCR showed that the titer of adeno-associated virus overexpressing HGF was 5.22×10^(12) vg·mL^(-1),and the relative expression content of HGF protein in overexpression group was significantly higher than that in saline group or negative control group(P all<0.01).Conclusion In this study,HGF adeno-associated virus vector was successfully constructed and the virus titer reached the requirements of ordinary animal injection,which proved that the adeno-associated viral vector was successfully constructed.