中国组织工程研究Issue(49)：7995-8000,6.DOI:10.3969/j.issn.2095-4344.2014.49.022
构建人基质金属蛋白酶1基因重组腺病毒载体及体外降解Ⅲ型胶原的检测
Construction of recombinant adenovirus vector for human matrix metalloproteinase-1 gene and detection of collagen type III degradation in vitro
杜超 1蒋明德 1曾维政 1郑淑梅1
作者信息
- 1. 解放军成都军区总医院消化内科，四川省成都市 610083
- 折叠
摘要
Abstract
BACKGROUND:Matrix metaloproteinase-1 can degrade extracelular matrix, which is mainly colagen type I, and has the potential to reverse fibrosis tissue. OBJECTIVE:To construct the recombinant adenovirus vector containing human matrix metaloproteinase-1 (hMMP-1) gene with GatewayTM Clone Technology, and observe the capacity of degrading colagen type IIIin vitro. METHODS: The gene hMMP-1 was amplified by using PCR from the pcDNA3.1 plasmid and was cut down by the double endonuclease. The linear gene fragment was connected to the entry vector pENTERTM 1A. Then the entry clone and the destination vectors pJTI™ R4 Dest CMV-N-EmGFP pA Vector recombined using the LR reaction to form the expression clone pAd-hMMP-1-eGFP. The linear pAd-hMMP-1-eGFP cut down by endonucleasePac I was transfected into HEK293A cels to packaging the Ad-hMMP-1-eGFP. The transfected situation was observed under a fluorescence microscope, the target protein expression was detected by western-blot assay and RT-PCR. Cels can be divided into three groups: blank control group: HEK293A cels, AD-EGFP group: HEK293A cels were infected by Ad-eGFP, AD-HMMP1-EGF group: HEK293A cels were infected by Ad-hMMP1-eGFP and colagen type III. The content of colagen type III was detected by ELISA kits after 24, 48 and 72 hours. RESULTS AND CONCLUSION: It was confirmed that the entry vector and the destination vector both contained hMMP-1 target gene by restriction analysis and sequencing. The green fluorescent protein was observed in the 293A cels transfected by the Ad-hMMP-1-eGFP at 4 days. The fluorescence intensity was the highest at 10 days. The virus was colected at 12 days, the viral titer was determined as 4.84 × 1010 PFU/mL, the target protein was efficient expressionvia western-blot assay. Blank control group and AD-EGFP group had no obvious change of colagen content with the extension of time. The rate of colagen degradation in AD-HMMP1-EGFP group was 24%, 56% and 81% respectively at 24, 48, 72 hours. AD-HMMP1-EGFP group degraded colagen significantly compared with the other two groups (P < 0.01). The recombinant adenovirus vector containing hMMP-1 was successfuly constructed by using the Gateway technology, this method was more efficient and specific than with the traditional methods. The hMMP1 degraded colagen type III significantlyin vitro.关键词
实验动物/组织工程/人基质金属蛋白酶1基因/Gateway技术/重组腺病毒载体/Ⅲ型胶原Key words
matrix metaloproteinase-1/virus/colagen type III分类
医药卫生引用本文复制引用
杜超,蒋明德,曾维政,郑淑梅..构建人基质金属蛋白酶1基因重组腺病毒载体及体外降解Ⅲ型胶原的检测[J].中国组织工程研究,2014,(49):7995-8000,6.
