| 摘要: |
| 目的 构建并表达野生型及突变型肾上腺脑白质营养不良蛋白（adrenoleukodystrophy protein，ALDP）的慢病毒载体，探讨ABCD1基因突变对ALDP结构和功能的影响。方法 选择ABCD1基因的H283R和P534R两个突变，首先采用生物信息学方法，进行突变致病性及突变体结构稳定性预测；再利用分子克隆技术，将ABCD1基因克隆到pLEX-MCS慢病毒载体，构建野生型慢病毒载体：pLEX-ABCD1，定点诱变构建2个突变型重组载体：pLEX-ABCD1-H283R和pLEX-ABCD1-P534R，并与其他包装载体共转染293T细胞包装病毒。收集病毒并感染宿主细胞，RT-PCR检测慢病毒感染细胞中野生型与突变型ABCD1 mRNA表达，免疫荧光及蛋白质免疫印迹法分析野生型与突变型ALDP亚细胞定位及表达。结果 生物信息学预测显示H283R和P534R为ALD致病性突变；RT-PCR结果显示慢病毒感染细胞中野生型与突变型ABCD1 mRNA均过表达；免疫荧光及蛋白质免疫印迹结果表明，H283R和P534R突变可能导致ALDP突变体表达量下降，但未观察到ALDP定位改变。结论 成功构建ABCD1基因慢病毒表达载体，并评估了H283R和P534R突变对ALDP表达及亚细胞定位的影响，为深入研究ALD发病机制提供了实验依据。 |
| 关键词: ABCD1基因 肾上腺脑白质营养不良蛋白 计算生物学 慢病毒属 |
| DOI：10.3724/SP.J.1008.2014.00884 |
| 投稿时间：2013-12-09修订日期：2014-06-03 |
| 基金项目:国家自然科学基金青年基金（31200932），福建省自然科学基金青年创新项目（2012J05158）. |
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| Construction and expression of lentiviral vector containing adrenoleukodystrophy gene |
| ZHANG Lin,FU Xian-guo,LIN Yu-xiang,LAN Feng-hua,WANG Zhi-hong* |
| (Department of Experimental Medicine, Affiliated Dongfang Hospital, Xiamen University(Fuzhou General Hospital, PLA Nanjing Military Area Command), Fuzhou 350025, Fujian, China) |
| 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. |
| Key words: ABCD1 gene adrenoleukodystrophy protein computational biology lentivirus |
