白细胞介素-10腺病毒载体制备及其在血管平滑肌细胞的表达

Construction of Adenovirus Vector with Human Interleukin 10 and Its Expression in Vascular Smooth Muscle Cells

目的构建人白细胞介素(human interleukin,hIL)-10和绿色荧光蛋白(green fluorescent protein,GFP)双表达腺病毒载体,观察目的基因在血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的表达。方法以T载体(pUCm-T/hIL-10cDNA)为模板,PCR扩增hIL-10cDNA,连入穿梭质粒,卡那霉素抗性筛选,NotⅠ、XholⅠ酶切鉴定,测序,局部序列搜索工具(BLAST)分析序列正确,PmeⅠ酶切,电穿孔单独转化BJ5183-AD-1,转化XL10-Gold扩增,PacⅠ酶切,AD-293细胞包装,PCR鉴定,测定病毒滴度,脂质体介导转染VSMCs,荧光显微镜下观察GFP的表达,酶联免疫吸附试验(ELISA)检测培养上清液中hIL-10的含量。结果重组穿梭质粒构建正确,同源重组后PacⅠ酶切获得30kb和3kb片段,重组病毒滴度为3×1010efu/ml,PCR检测含目的基因,转染的VSMCs可见绿色荧光,hIL-10含量为25ng/106个细胞。结论构建的双表达载体于VSMCs成功表达,为血管内膜增生的基因治疗打下基础。

Objective To construct recombinant adenovirus vector co-expressing human interleukin （hIL）-10 and green fluorescent protein （GFP） for study of the expression of genes of interest in vascular smooth muscle cells （VSMCs）. Methods hIL-10 cDNA was amplified from pUCm-T/hIL-10 cDNA using polymerase chain reaction （PCR）, and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1. Kanamycin resistance screeninged for recombinant plasmids, which were linealized with Pine Ⅰ and transformed into BJ5183-AI）-Ⅰ containing pAdEasy-1 by electropo ration after determining the insert＇s sequence correct by Not Ⅰ and Xhol Ⅰ digestion, sequencing and basic local alignment search tool （BLAST）. Prepared recombinant adenovirus plasmids were transformed into XL10-Gold cells. Amplified plasmids were transfected to AI）-293 cells for packaging after being linearized with Pac Ⅰ. PCR was used to determine target gene; The titer of the recombinant adenovirus was measured. VSMCs were transfected by recombinant adenovirus and viewed under fluorescence microscope, hIL-10 concentration in transfected VSMCs supernant was measured by enzyme linked immune sorbent assay （ELISA）. Results Recombinant shuttle plasmids contained interest gene. Recombinant adenovirus had 30 kb and 3 kb fragments after digestion with Pac Ⅰ . PCR indicated that the recombinant adenovirus contained interest gene. The titer of recombinant adenovirus was 3 × 10^10 efu/ml. Transfected VSMCs had GFP expression and hIL-10 concentration in supernatant was 25 ng/10^6 cells. Conclusion The recombinant adenovirus co-expressing hIL-10 and GFP is successfully constructed and could effectively express in VSMCs, this lays the foundation for the gene therapy of vascular intimal hyperplasia.