[1]杜先智,周向东.天然抗菌多肽elafin重组腺病毒载体构建及气道上皮细胞表达[J].第三军医大学学报,2007,29(11):1042-1045.
 DU Xian-zhi,ZHOU Xiang-dong.Construction of recombinant adenovirus vector Ad-elafin for nature antibiotic elafin gene and its expression in primary airway epithelium[J].J Third Mil Med Univ,2007,29(11):1042-1045.
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天然抗菌多肽elafin重组腺病毒载体构建及气道上皮细胞表达
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第11期
页码:
1042-1045
栏目:
论著
出版日期:
2007-06-15

文章信息/Info

Title:
Construction of recombinant adenovirus vector Ad-elafin for nature antibiotic elafin gene and its expression in primary airway epithelium
作者:
杜先智 周向东
重庆医科大学附属第二医院呼吸内科
Author(s):
DU Xian-zhi ZHOU Xiang-dong
Department of Respiratory Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
关键词:
elafin腺病毒气道上皮细胞
Keywords:
elafin adenovirus airway epithelium
分类号:
R322.35; R341.6; R394-33
文献标志码:
A
摘要:
目的    构建天然抗菌多肽elafin基因高效表达载体——重组腺病毒载体,并探讨其在肺上皮细胞中的初步表达。    方法    提取人肺总RNA,进行elafin基因逆转录多聚酶链反应(RT-PCR)扩增,PCR产物双酶切后亚克隆至穿梭质粒pAdTrack-CMV上;在BJ5183细菌内和pAdEasy-1同源重组,筛选阳性克隆,酶切、PCR及测序鉴定,PacⅠ酶切线性化后脂质体法转染293细胞进行包装,获得腺病毒载体Ad-elafin;继之在293细胞内扩增,利用报告基因荧光蛋白(GFP)监测病毒滴度和感染效率;重组腺病毒Ad-elafin转染原代培养的气道上皮细胞,24 h后荧光显微镜观察GFP表达,酶联免疫吸附测定法(ELISA)检测培养上清液中elafin的含量。    结果    测序、酶切及PCR证实天然抗菌多肽elafin基因重组腺病毒载体Ad-elafin构建成功,同时转染气道上皮细胞24 h后荧光显微镜可见GFP的表达,且转染组细胞上清液中elafin测定值为(2.4±0.4)ng/ml与对照组(1.9±0.3)ng/ml相比,差异有统计学意义(P<0.05)。    结论    elafin基因重组腺病毒载体成功构建,有利于elafin生物特性的研究。
Abstract:
Objective    To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.      Methods    The elafin gene was amplified by RT-PCR from lung tissues, then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and sequencing. Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification. Infection titer and rate were monitored by green fluorescent protein (GFP) expression. Finally the primary airway epithelium was infected with Ad-elafin, and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection. The elafin protein was detected by ELISA in the supernatant.     Results    Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully. Twenty-four hours after transfection, the GFP expression was seen by fluorescence microscopy. The concentration of elafin protein was (2.2±0.4) ng/ml in supernatant of transfected group, while that of control group was (1.9±0.3) ng/ml, P<0.05.     Conclusion    The recombinant adenovirus vector Ad-elafin was constructed successfully  and can be helpful to research the characteristic of elafin.

参考文献/References:

杜先智, 周向东. 天然抗菌多肽elafin重组腺病毒载体构建及气道上皮细胞表达[J]. 第三军医大学学报, 2007, 29(11):1042-1045.

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更新日期/Last Update: 2008-10-23