CircKat6b重组腺相关病毒制备及其在小鼠海马中的表达

Construction of circKat6b recombinant adeno-associated virus and its expression in the hippocampus of mice

目的获得表达circKat6b的重组腺相关病毒(recombinant adeno-associated virus expressing circKat6b,rAVV-circKat6b),检测其在小鼠海马组织中的感染效率和表达水平。方法将mmu-circKat6b基因片段插入腺相关病毒(adeno-associated virus,AAV)穿梭质粒中构建重组AAV质粒,将重组AAV质粒、包装质粒和辅助质粒通过脂质体LipoFiter^(TM)共同转染293T细胞包装rAAV-circKat6b。从转染的293T细胞中收集并通过柱纯化rAAV-circKat6b,采用实时荧光定量聚合酶链反应(qRT-PCR)测定rAAV-circKat6b滴度。最后通过小鼠脑立体定位仪注射rAAV-circKat6b至小鼠海马组织中,1个月后免疫荧光显微镜观察rAAV-circKat6b的感染效率,qRT-PCR检测rAAV-circKat6b表达水平。结果包装成功的rAAV-circKat6b经过纯化后获得了高滴度的rAAV-circKat6b,qRT-PCR检测rAAV-circKat6b的病毒滴度达到1.2×10^(12) vg/mL。感染1个月后,脑组织海马区域绿色荧光表达明显,circKat6b表达水平明显增加。结论成功构建的rAAV-circKat6b可使小鼠海马组织中circKat6b高表达,这一研究结果为进一步研究circKat6b的生物学功能提供了实验基础。

Objective Recombinant adeno-associated virus expressing circKat6b(rAVV-circKat6b)was obtained and its infection effect and expression level of circKat6b in the hippocampus of mice were examined.Methods The mmu-circKat6b gene fragment was inserted into AAV expression plasmid to construct a recombinant AAV plasmid.The recombinant AAV plasmid,packaging plasmid and helper plasmid were co-transfected into 293T cells by Lipofiter^(TM) for rAAV-circKat6b packaging.The rAAV-circKat6b was collected from transfected 293T cells and purified by column,and the titer of the rAAV-circKat6b was tested by qRT-PCR.Finally,rAAV-circKat6b was injected into the hippocampus of mice by stereotactic apparatus.One month later,the infection effect of rAAVcircKat6b was observed by immunofluorescence microscope,and the expression level of rAAV-circKat6b was detected by qRT-PCR.Results The successfully packaged rAAV-circKat6b was purified to obtain high titer rAAV-circKat6b,and the virus titer of rAAVcircKat6b was detected by qRT-PCR and reached 1.2×10^(12) vg/mL.Strong green fluorescence and high level of circKat6b were observed in the infected hippocampus after one month of infection.Conclusions The successfully constructed rAAV-circKat6b could highly increase the expression level of circKat6b in the hippocampus of mice,which provided an effective tool for further studying of the biological functions of circKat6b.