Construction and identification of recombinant adenovirus vectors carrying the human transcription factor PU.1 gene
10.16571/j.cnki.1008-8199.2016.05.004
- VernacularTitle:人转录因子 PU ．1基因重组腺病毒载体的构建及鉴定
- Author:
Chenyang LIU
;
Wenjie YAN
;
Min WANG
;
Wenkui SUN
;
Xin SU
;
Yi SHI
- Publication Type:Journal Article
- Keywords:
PU.1;
SPI1;
Recombinant adenovirus;
Asper-gillus fumigatus;
Innate immunity
- From:
Journal of Medical Postgraduates
2016;29(5):465-469
- CountryChina
- Language:Chinese
-
Abstract:
Objective PU.1 plays a key role in innate immune function in the alveolar macrophage.This study was to con-struct and identify recombinant adenovirus vectors carrying the human transcription factor PU.1 gene. Methods The recombinant shut-tle plasmid was obtained from the PU.1 gene ( SPI1) and eukaryotic expression vector that digested by restriction enzymes and connected by T4 DNA ligase.The target fragment SPI1-IRES-EGFP was amplified by PCR.The product was cloned into the intermediate pDONR221 and then recombined with the adenovirus backbone plasmid pAd/CMV/V5-DEST to form a recombinant adenovirus vector. The recombinant adenovirus vector was linearized by PacI and then transfected into human embryonic kidney (HEK293) cells to obtain the recombinant adenovirus pAD-SPI1-IRES-EGFP, which was then propagated in HEK293 cells, filtered and purified to obtain high-con-centration adenoviruses.The adenovirus titer was determined by TCID 50 assay.The PU.1 gene expression in the HEK293 cells was con-firmed by fluorescence microscopy and real-time qPCR. Results PCR amplification, restriction digestion and sequencing analysis showed the recombinant adenovirus carried the correct PU.1 gene.The final virus titer, calculated by TCID 50, was 8 ×1011 IU/mL. Green fluorescence was observed under the fluorescence microscope. Real-time qPCR confirmed that the expression of PU.1 mRNA was increased by 2189.93 folds. Conclusion The recombinant adenovirus vector carrying the PU.1 gene was constructed and obtained successfully, which could contribute to further studies of the influence of PU.1 overex-pression on the innate defense against Aspergillus fumigatus.
