Construction of recombinant adenovirus vector for human matrix metalloproteinase-1 gene and detection of collagen type III degradation in vitro

doi:10.3969/j.issn.2095-4344.2014.49.022

Abstract

Abstract:

BACKGROUND: Matrix metalloproteinase-1 can degrade extracellular matrix, which is mainly collagen type I, and has the potential to reverse fibrosis tissue.

OBJECTIVE: To construct the recombinant adenovirus vector containing human matrix metalloproteinase-1 (hMMP-1) gene with GatewayTM Clone Technology, and observe the capacity of degrading collagen type III in 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 pENTER^TM 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 endonuclease Pac I was transfected into HEK293A cells 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. Cells can be divided into three groups: blank control group: HEK293A cells, AD-EGFP group: HEK293A cells were infected by Ad-eGFP, AD-HMMP1-EGF group: HEK293A cells were infected by Ad-hMMP1-eGFP and collagen type III. The content of collagen 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 cells transfected by the Ad-hMMP-1-eGFP at 4 days. The fluorescence intensity was the highest at 10 days. The virus was collected at 12 days, the viral titer was determined as 4.84 × 10¹⁰ PFU/mL, the target protein was efficient expression via western-blot assay. Blank control group and AD-EGFP group had no obvious change of collagen content with the extension of time. The rate of collagen degradation in AD-HMMP1-EGFP group was 24%, 56% and 81% respectively at 24, 48, 72 hours. AD-HMMP1-EGFP group degraded collagen significantly compared with the other two groups (P < 0.01). The recombinant adenovirus vector containing hMMP-1 was successfully constructed by using the Gateway technology, this method was more efficient and specific than with the traditional methods. The hMMP1 degraded collagen type III significantly in vitro.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: matrix metalloproteinase-1, virus, collagen type III

CLC Number:

R318

Du Chao, Jiang Ming-de, Zeng Wei-zheng, Zheng Shu-mei. Construction of recombinant adenovirus vector for human matrix metalloproteinase-1 gene and detection of collagen type III degradation in vitro [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(49): 7995-8000.

Figures/Tables 1

2.1 目的基因的获取及测序结果从携带人基质金属蛋白酶1的pcDNA3.1质粒中PCR扩增出约1 428 bp大小基因条带(图1)，经测序鉴定所获序列与GenBank报道序列完全一致。 2.2 入门克隆的构建及鉴定结果经限制性内切酶 EcoⅠ和XhoⅠ双酶切基因片段人基质金属蛋白酶1和约3 891 bp的pENTERTM 1A质粒后，成功连接构建入门克隆质粒pENTERTM 1A-hMMP-1，其中第4泳道为阳性结果，双酶切后产物电泳条带大小分别为1 417 bp和3 891 bp，测序结果显示与理论完全相符(图2)。R 2.3 表达克隆的构建及鉴定结果利用LR反应体外重组入门克隆pENTERTM 1A-hMMP-1和目的载体pJTITM R4 Dest CMV-N-EmGFP pA Vector后，转化入感受态大肠杆菌Stbl3，挑选阳性菌落进行PCR鉴定，引物为pAd- hMMP-1-f：GAA CCC ACT GCT TAC TGG CTT；pAd- hMMP-1-r：TCG AGA CCG AGG AGA GGG T；产物大小2 951 bp；与第3泳道凝胶电泳条带结果相符，送交测序鉴定结果完全正确(图3)。 2.4 重组腺病毒包装及鉴定结果经PacⅠ酶切线性化腺病毒质粒pAd- hMMP-1-eGFP并转入HEK293A细胞后，4 d左右在荧光显微镜下可观察到绿色荧光蛋白表达，10 d左右荧光强度达到最高(图4)。将收集到的目的蛋白进行Western blot鉴定，结果证实人基质金属蛋白酶1蛋白在重组腺病毒中高效表达(图5)。 2.5 腺病毒滴度测定(TCID50)结果将腺病毒按梯度浓度感染96孔板中HEK293细胞后，5 d左右细胞开始出现病变状态CPE(Cytopathic effect)，逐渐皱缩变小，形态不规则，并且细胞间缝隙增大，由贴壁状态变为悬浮，10 d后计数出现CPE的孔数(表1)，用Reed-Muench两氏法计算病毒滴度。 lgTCID50=距离比例×稀释度对数之间的差+高于50%病变率的稀释度的对数，即TCID50=10-9.84/0.1 mL，所得病毒滴度=4.84×1010 PFU/mL。 2.6 ELISA试剂盒检测人基质金属蛋白酶1体外降解Ⅲ型胶原蛋白能力细胞培养24，48，72 h后，收集含胶原蛋白细胞培养基上清，离心后用Ⅲ型胶原蛋白ELISA检测试剂盒分别进行检测，结果显示空白对照组、AD-EGFP组中胶原蛋白含量无明显变化，而AD-HMMP1-EGFP组中胶原蛋白降解率显著高于对照组(P < 0.01)，并且随时间延长胶原蛋白降解率在逐渐增加(表2)。 "

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