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

作　　者：杜超[1] 蒋明德[1] 曾维政[1] 郑淑梅[1] 

机构地区：[1]解放军成都军区总医院消化内科,四川省成都市610083

出　　处：《中国组织工程研究》2014年第49期7995-8000,共6页Chinese Journal of Tissue Engineering Research

摘　　要：背景:基质金属蛋白酶1能够降解以Ⅰ型胶原为主的细胞外基质,有望逆转纤维化组织。目的:利用Gateway技术构建含人基质金属蛋白酶1基因的重组腺病毒载体,并观察其在体外降解Ⅲ型胶原蛋白的能力。方法:从携带人基质金属蛋白酶1基因片段的pc DNA3.1质粒中PCR扩增出人基质金属蛋白酶1基因片段,双酶切连接入门载体p ENTERTM 1A形成入门克隆,利用LR反应与目的载体p JTI TM R4 Dest CMV-N-Em GFP p A Vector同源重组构建表达克隆p Ad-h MMP-1-e GFP。经PacⅠ酶切线性化转入HEK293A细胞包装获得重组腺病毒Ad-h MMP-1-e GFP,RT-PCR及Western-Blot法分别检测目的基因与蛋白表达。细胞分为3组:1空白对照组:HEK293A细胞。2AD-EGFP组:Ad-e GFP感染HEK293空白对照组。3AD-HMMP1-EGFP组:Ad-h MMP-1-e GFP感染HEK293空白对照组。分别加入相同含量Ⅲ型胶原蛋白,在24,48,72 h用ELISA试剂盒检测Ⅲ型胶原蛋白水平。结果与结论:经酶切及测序鉴定证实入门载体和目的载体中均含有人基质金属蛋白酶1目的基因。重组腺病毒Ad-h MMP-1-e GFP感染293A细胞后4 d观察到绿色荧光表达,10 d荧光强度达最高,12d收集病毒液;测定病毒滴度为4.84×1010PFU/m L,Western-Blot法检测目的蛋白高效表达。随时间延长,空白对照组和AD-EGFP组胶原蛋白含量无明显变化,AD-HMMP1-EGFP组24,48,72 h胶原蛋白降解率分别为24%,56%,81%,与空白对照组、AD-EGFP组相比AD-HMMP1-EGFP组降解胶原效果显著(P<0.01)。利用Gateway技术成功构建含人基质金属蛋白酶1基因的重组腺病毒载体,与传统构建方法相比具有高效性和特异性,分泌基质金属蛋白酶1检测其降解Ⅲ型胶原蛋白效果显著。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^TM 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＆amp;nbsp;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 ＆#215; 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 gr

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 

分 类 号：R318[医药卫生—生物医学工程]