Construction and expression of lentiviral vector containing adrenoleukodystrophy gene
10.3724/SP.J.1008.2014.00884
- Author:
Lin ZHANG
1
Author Information
1. Department of Experimental Medicine, Affiliated Dongfang Hospital, Xiamen University(Fuzhou General Hospital, PLA Nanjing Military Area Command)
- Publication Type:Journal Article
- Keywords:
ABCD1 gene;
Adrenoleukodystrophy protein;
Computational biology;
Lentivirus
- From:
Academic Journal of Second Military Medical University
2014;35(8):884-891
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a lentiviral vector carrying the wild-type and mutant adrenoleukodystrophy gene(ABCD1) and to investigate the effects of ABCD1 mutation on the structure and function of adrenoleukodystrophy protein(ALDP). Methods Different computational algorithms were used to predict the pathogenicity and the structural stability of ALDP mutants: H283R and P534R. Lentiviral vectors carrying wild type and mutants ABCD1 gene were constructed with pLEX-MCS, namely, pLEX-ABCD1, pLEX-ABCD1-H283R and pLEX-ABCD1-P534R. The recombinant plasmids and two packaging vectors were co-transfected into 293T cells to obtain virus, and the latter was used to infect host cells. The expression of the wild type and mutant ABCD1 mRNA in lentivirus infected cells was detected by RT-PCR. The subcellular localization and expression of the wild type and mutant ALDP were detected by immunofluorescence and Western blotting analysis. Results Bioinformatic prediction results showed that both mutations in this study were at conserved codons, suggesting a pathogenic nature. Overexpression of the wild type and mutant ABCD1 mRNA was detected by RT-PCR in lentivirus infected cells. Immunofluorescence study and Western blotting analysis showed overexpression of the wild type ALDP and lower expression of the mutant ALDP, with no subcellular mislocalization of the mutant ALDP detected. Conclusion We have successfully constructed a recombinant lentiviral vector carrying the ABCD1 gene and assessed the effects of the ABCD1 mutations on the expression and localization of ALDP, providing evidence for understanding the pathogenic mechanism of ALD.
