PPENK-MIDGE-NLS基因载体的构建及其在活体大鼠的表达

Construction of PPENK-MIDGE-NLS gene vector and the expression in rat

摘要:

增加体内免疫细胞合成分泌内源性阿片肽，可对心肌缺血再灌注损伤产生保护作用。基因治疗是增加内源性脑啡肽 (Enkephalin, ENK) 的一种有前景的研究方向，但是以往的病毒、质粒等载体受到其自身免疫原性的限制并存在基因重组、原癌基因激活、抗细菌蛋白抗体产生以及基因表达改变等诸多问题。本研究采用非病毒、非质粒的微量免疫原定义的基因表达 (Minimalistic immunological defined gene expression, MIDGE) 方法构建前脑啡肽原 (Preproenkephalin, PPENK) PPENK-MIDGE-NLS基因载体能克服病毒和质粒载体的上述缺点。用PCR技术扩增大鼠前脑啡肽原 (Preproenkephalin, PPENK) 外显基因，产物插入pEGFP-N1质粒，双酶切质粒获得包含启动子、目的基因、RNA稳定序列的线性载体，两端以不受外切酶作用的发夹样脱氧寡核苷酸序列 (Oligodesoxynucleotides, ODNs) 封闭。为保证载体的入核和表达效率，载体的一端连接了核定位序列 (Nuclear localization sequence, NLS)。流式细胞术和激光共聚焦检测其转染效率，Western blotting检测组织内前脑啡肽蛋白的表达。结果显示，PPENK-MIDGE-NLS能够转染大鼠活体细胞并表达前脑啡肽蛋白，增加载体的量能够在一定范围内提高转染效率。研究结果表明该载体可能成为预防和治疗心肌缺血再灌注损伤的新 方法。

Abstract:

Increasing the production and secretion of endogenous opioid peptide by immune cell can significantly induce myocardial protective effects against ischemia-reperfusion injury. Gene therapy is promising to increase endogenous enkephalin (ENK). However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, gene recombination, oncogene activation, the production of antibacterial antibody and changes in physiological gene expression. Minimalistic immunologically defined gene expression (MIDGE) can overcome all the deficients of viral and plasmid vectors. The exon of rat’s preproenkephalin (PPENK) gene was amplified by PCR and the fragments were cloned into pEGFP-N1 plasmids. The recombined plasmids were digested with enzymes to obtain a linear vector contained promoter, preproenkephalin gene, RNA stable sequences and oligodesoxy nucleotides (ODNs) added to both ends of the gene vector to protect gene vector from exonuclease degradation. A nuclear localization sequence (NLS) was attached to an ODN to ensure the effective transport to the nucleus and transgene expression. Flow cytometry, laser confocal microscopy and Western blotting demonstrated that PPENK-MIDGE-NLS can transfect leukocyte of rat in vivo, increase the expression of proenkephalin (PENK) in tissue, and the transfection efficiency depends on gene vector’s dosage. These results indicate that PPENK-MIDGE-NLS could be an innovative method to protect and treatment of myocardial ischemia-reperfusion injury.