过表达OPTN和TAU-P301-L基因腺相关病毒构建与鉴定

Construction and identification of adeno-associated virus overexpressing OPTN and TAU-P301-L gene

目的获得能够过表达OPTN和TAU-P301-L基因的重组腺相关病毒,检测其在体外对293T细胞的感染,并且为阿尔茨海默氏病(AD)研究提供新思路。方法从含有目的基因的大肠杆菌中提取包涵目的基因的质粒,利用PCR扩增获得目的基因,目的基因OPTN构建到pAAV-IRES-ZsGreen1载体,目的基因TAU-P301L-V5构建到pAAV-CMV-mkate-IRES载体。鉴定质粒阳性克隆并测序。重组质粒pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5分别与包装质粒pAAV-RC9、辅助质粒pHelper通过Lipofiter^(TM)2000共转染293T细胞,获得AAV9病毒。利用相同方法,不使用目的基因构建对应绿色和红色AAV9对照病毒。qPCR法测定重组腺相关病毒载体滴度,病毒载体感染293T细胞进行病毒有效性及安全性鉴定,Western blotting和RT-PCR进行蛋白表达的鉴定,MTT检测OPTN蛋白与TAU-P301-L蛋白对293T细胞活力的影响。结果测序结果显示成功构建pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5腺相关病毒载体,qPCR测得病毒滴度为1.0×10^(12) vg/ml,病毒载体感染293T细胞转染效率为33.6%和20.5%, Western blot和RT-PCR显示蛋白过量表达。MTT结果显示OPTN组挽救了TAU-P301-L组的细胞凋亡。结论成功构建过表达OPTN和TAU-P301-L基因的腺相关病毒。OPTN蛋白可以减弱TAU-P301-L蛋白的细胞毒性。

Objective To obtain recombinant adeno-associated virus capable of over-expressing OPTN and TAU-P301-L genes,to detect its infection of 293 T cells in vitro,and to provide new ideas for the study of Alzheimer’s disease(AD).Methods The plasmid containing the gene of interest was extracted from Escherichia coli containing the gene of interest,and the target gene was obtained by PCR amplification.The target gene OPTN was constructed into pAAV-IRES-ZsGreen1 vector,and the target gene Tau-P301 L-V5 was constructed into pAAV-CMV-mkate-IRES vector.Plasmid positive clones were identified and sequenced.The recombinant plasmids pAAV-IRES-ZsGreen1-AAV9-OPTN and pAAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 were co-transfected into 293 T cells with the packaging plasmid pAAV-RC9 and the helper plasmid pHelper by LipofiterTM 2000,respectively,to obtain AAV9 virus.Using the same method,the corresponding green and red AAV9 control virus were constructed without using the target gene.The titer of recombinant adeno-associated virus was determined by qPCR method.The virulence and safety of 293 T cells were detected by viral vector,and the protein expression was identified by Western blot and RT-PCR.MTT assay was used to detect the effect of OPTN protein and TAU-P301-L protein on the activity of 293 T cells.Results The sequencing results showed that the AAV-IRES-ZsGreen1-AAV9-OPTN and pAAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 adeno-associated virus vectors were successfully constructed.The virus titer determined by qPCR was 1.0×1012 vg/ml.The transfection efficiency of virus vector-infected 293 T cells was 33.6%and 20.5%.Western blot and RT-PCR showed protein overexpression.MTT results showed that the OPTN group rescued the apoptosis of the TAU-P301-L group.Conclusion Adeno-associated virus over-expressing OPTN and TAU-P301-L genes was successfully constructed.OPTN protein can reduce the cytotoxicity of TAU-P301-L protein.