小鼠TFEB基因重组腺相关病毒的构建及鉴定

Construction and identification of mouse TFEB gene recombinant adeno-associated virus

目的构建小鼠TFEB基因的重组8型腺相关病毒(AAV8),并通过感染293细胞观察TFEB表达情况。方法将pUC57-mTFEB及载体质粒分别进行酶切、连接、转化,得到重组质粒pAAV-CMV-mTFEB,并进行PCR鉴定及测序;将鉴定正确重组质粒,进行AAV8病毒载体包装;正确包装病毒载体后,感染293细胞,通过Western blot观察TFEB基因在293细胞表达情况。结果酶切鉴定和测序结果证明了pAAV-CMV-mTFEB病毒包装成功;病毒滴度4.65×10^(12)vg/m L;细胞实验提示相比对照组,实验组TFEB基因表达水平升高6倍余(P<0.01)。结论 rAAV2/8-CMV-mTFEB构建及包装成功,并可以有效感染和表达。

Objective To construct the recombinant adeno-associated virus serotype 8（AAV8）of mouse TFEB gene and to observe its abi Iity to infect 293 cells. Methods The pUC57-m TFEB and the vector plasmid was digested respectively,connected and transformed to the recombinant plasmid pAAV-CMV-mTFEB. The recombinant plasmid was identified by po Iymerasechain reaction（PCR）and sequencing. The correct recombinant plasmid was packaged within AAV8. Then the recombinant adeno-associated virus of mouse TFEB gene was transfected into 293 ce IIs. After transfection,the expression level of target gene was analyzed by Western Blot. Results The success of TFEB gene inserted into the Adeno-associated virus vector was confirmed by restriction endonucIease and squencing. The titer of the recombinant adeno-associated virus was 4.65×10^（12）vg/m L. The expression of TFEB in experiment cells has increased over six-fold than that in the control（P〈0.01）. Conclusion The rAAV2/8-CMV-mTFEB was successfu IIy constructed and pakaged. Furthermore,it could transfected and expressed in cells effectively.