Construction and identification of a recombinant adenoviral vector expressing murine dendritic cell-associ-atedC-type lectin-1
- VernacularTitle:小鼠树突状细胞相关C型凝集素-1基因重组腺病毒载体的构建及鉴定
- Author:
Di XIA
;
Qian QIAN
;
Zhicheng LIU
;
Mingming TAN
;
Yuan DING
;
Xin SU
;
Wenkui SUN
;
Yi SHI
- Publication Type:Journal Article
- Keywords:
Dectin-1;
CLEC7A gene;
Adenovirus vector;
Pattern recognition receptor
- From:
Journal of Medical Postgraduates
2015;(4):341-345
- CountryChina
- Language:Chinese
-
Abstract:
Objective Dendritic cell-associatedC-type lectin-1 ( Dectin-1) is one of the most important receptors in antifungal innate immune response.This study was to construct a recombinant adenovirus vector expressing themurine Dectin-1gene and acquire a high-concentration adenovirus by amplification and purification. Methods The PCR amplification product CLEC7A-pIRES2-EGFP was cloned into the intermediate vector pDONR221, and then recom-bined with the backbone vector pAD/CMV/V5-DEST to produce a re-combinant plasmid pAD-CLEC7A-pIRE2S -EGFP.The recombinant plasmid was linearized with Pac I and transfected into human embryon-ic kidney ( HEK293) cells to produce recombinant adenovirus pAD-CLEC7Ap-IRES 2-EGFP. The adenovirus was propagated in the HEK293 cells and purified by filtering through the cellulose acetate membrane and concentrating column.Fluorescence microscopy and re-al-time PCR were used to determine the expression of the Dectin-1 gene. Results PCR identification, enzyme digestion, and sequen-cing results manifested theDectin-1 gene in the vector, with the final adenovirus titer of 5×1011 IU/mL.Fluorescence microscopy revealed green fluorescence and real-time PCR assay confirmed that the expression of Dectin-1 was improved by 8677.25 times. Conclusion A relatively high-titer adenovirus expressing Dectin-1 was acquired,which may help to further study the high expression of Dectin-1 in anti-fungal innate immunity in vitro and in vivo.
