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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 adenovirus-8 (AAV8) gene of mouse TFEB gene and observe the expression of TFEB by infecting 293 cells. Methods The recombinant plasmids pAAV-CMV-mTFEB were digested, ligated and transformed into pAAV-CMV-mTFEB. The recombinant plasmid pAAV-CMV-mTFEB was identified and sequenced. The correct recombinant plasmid was identified and packaged in AAV8 viral vector. , Infected 293 cells, the expression of TFEB gene in 293 cells was observed by Western blot. Results The restriction endonuclease digestion and sequencing proved that the packaging of pAAV-CMV-mTFEB virus was successful. The virus titer was 4.65 × 10 ~ (12) vg / m L. The results of cell experiments showed that compared with the control group, the TFEB gene expression level increased 6 More than (P <0.01). Conclusion The construction and packaging of rAAV2 / 8-CMV-mTFEB was successful and could be effectively infected and expressed.