慢病毒载体介导线虫sfat-1基因转化牛胎儿成纤维细胞优化脂肪酸组份的研究
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摘要
多不饱和脂肪酸(Polyunsaturated Fatty Acids,PUFAs)是指化学结构中含有两个或两个以上不饱和烯键的长链脂肪酸。近年来随着对PUFAs生理学作用和细胞学功能研究的深入以及在医药学领域和营养学领域应用的开发, PUFAs已经成为脂肪酸研究领域中新兴的热点。ω-6 PUFAs和ω-3 PUFAs是PUFAs家族中最主要的成员,它们在机体的新陈代谢、信号转导及免疫调节等生理活动中起着非常重要的作用。ω-3脂肪酸脱氢酶(ω-3 Fatty Acid Desaturase)是存在于极少数动植物体内、以ω-6 PUFAs为底物催化生成相应ω-3 PUFAs的一种去饱和酶,编码这种酶的基因称为ω-3脂肪酸脱氢酶基因(ω-3 Fatty Acid Desaturase Gene)。
本实验使用密码子替换法优化线虫Caenorhabditis.briggsae的ω-3脂肪酸脱氢酶基因sfat-1并用化学合成法在体外合成该基因。利用两端含有重叠碱基序列的若干个寡核苷酸短片段和相应引物在PCR体系中连接扩增,再经过酶切连接进而合成总长度为1200bp的目的片段。将化学合成的sfat-1基因和表达载体pcDNA3.1(-)进行连接后转染大肠杆菌感受态细胞DH5α,经LB平板筛选阳性克隆、提取质粒和酶切鉴定后进行测序用以验证目的基因的克隆。
将ω-3脂肪酸脱氢酶基因sfat-1连接于慢病毒载体共转染HEK293T细胞构建携带目的基因的重组慢病毒pGCLV-sfat1-GFP,同时构建空白慢病毒pGCLV-GFP为实验对照组。慢病毒载体经过酶切、连接和包装后进行收集、纯化浓缩、滴度测定及生物安全性检测。
用所获得重组慢病毒pGCLV-sfat1-GFP和慢病毒pGCLV-GFP转化中国西门塔尔牛胎儿成纤维细胞并测定慢病毒最佳MOI,观察并检测牛胎儿成纤维细胞内绿色荧光蛋白(GFP)基因的表达随细胞培养时间变化,提取细胞总RNA进行RT-PCR验证sfat-1基因的表达,使用重组慢病毒感染细胞后对细胞脂肪酸进行提取和成分分析。
实验结果表明:使用密码子替换法和PCR化学合成法能够在体外合成线虫Caenorhabditis.elegans的ω-3脂肪酸脱氢酶基因sfat-1;通过使用携带绿色荧光蛋白基因的慢病毒载体介导线虫sfat-1基因感染中国西门塔尔牛胎儿成纤维细胞能够稳定且高效地表达融合蛋白sfat1-GFP;由sfat-1基因编码的ω-3脂肪酸脱氢酶在牛胎儿成纤维细胞中能够有效地催化多种ω-6 PUFAs转变为相应的ω-3 PUFAs,使二者比例由原来的15.32:1变为0.83:1,并且在牛胎儿成纤维细胞中产生更加丰富的超长链ω-3 PUFAs。
本研究阐述了优化牛胎儿成纤维细胞内脂肪酸成分的有效方法,证明了优化后的线虫ω-3脂肪酸脱氢酶基因sfat-1在动物体细胞内的作用,为在实验和临床研究中制备转基因动物提供了一定的依据。
From epidemiology to cell culture and animal studies to randomized controlled trials, the cardioprotective effects of polyunsaturated fatty acids (PUFAs) are becoming recognized. Recently, Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have been the subject of increasing investigation and have attracted considerable interest as pharmaceutical and nutraceutical compounds. They are subtribe PUFAs and essential components requered for normal cellular function and have been shown to exert many preverntive and therapeutic actions.
An important class of enzymes involved in the synthesis of PUFAs is the fatty acid desaturases, which catalyze the introduction of double bonds into the hydrocarbon chain at a position determined by the enzyme specificity.The Caenorhabditis.briggsae sfat-1 gene, which is absent in most animals, encodes anω-3 fatty acid desaturase that convertsω-6 PUFAs toω-3 PUFAs. The objection of current study was the first attempt to transfer sfat-1 gene with lentivirus vectors in the aim to generate transgenic bovine fetal fibroblasts capable of convertingω-6 PUFAs toω-3 PUFAs.
To heterologously express the n-3 fatty acid desaturase in early bovine fetal fibroblasts, the sfat-1 gene encoding this protein by optimization of codon usage for mammalian cells was modified and was coupled to a pCMV enhancer/chickenβ-actin promoter (which allows high-level and broad expression of the transgenic in all serieses of bovine fibroblasts). The full length sfat-1 gene was constructed by chemical synthesis in vitro by PCR amplification and restriction ligation.
Lentiviral expression plasmid vectors containing GFP domains carrying the sfat-1 gene fragments (pGCLV-sfat1-GFP group) were constructed. Lentivirus with GFP (pGCLV-GFP gourp) domains was performed as a control. After sfat-1 gene fragment transfered, pGCLV-sfat1-GFP group cells and pGCLV-GFP group cells were examined via green fluorescent protein detection. RT-PCR and Western blotting were performed to identify the fusion protein. GC/MS was conducted to analysis the composition of fatty acid by using the extracted total cellular lipids.
Lentivirus-mediate sfat-1 had alterated the composition of fatty acid in Chinese Simmental bovine fetal fibroblasts significantly. Heterologous expression of the sfat-1 gene in bovine fetal fibroblasts capable of converting variousω-6 PUFAs to the correspondingω-3 PUFAs, and changed the n-6/n-3 ratio from about 15.32:1 to 0.83:1.
This study has demonstrated clearly that the sfat-1 gene can be expressed functionally in bovine fetla fibroblasts, and its expression could confer cells’capability of convertingω-6 PUFAs to correspondingω-3 PUFAs, leading to a balanced n-6/n-3 ratio and a change in fatty acids production. The rearch provides an effective approach to modifying fatty acid composition of bovine fetal fibroblasts and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.
本实验使用密码子替换法优化线虫Caenorhabditis.briggsae的ω-3脂肪酸脱氢酶基因sfat-1并用化学合成法在体外合成该基因。利用两端含有重叠碱基序列的若干个寡核苷酸短片段和相应引物在PCR体系中连接扩增,再经过酶切连接进而合成总长度为1200bp的目的片段。将化学合成的sfat-1基因和表达载体pcDNA3.1(-)进行连接后转染大肠杆菌感受态细胞DH5α,经LB平板筛选阳性克隆、提取质粒和酶切鉴定后进行测序用以验证目的基因的克隆。
将ω-3脂肪酸脱氢酶基因sfat-1连接于慢病毒载体共转染HEK293T细胞构建携带目的基因的重组慢病毒pGCLV-sfat1-GFP,同时构建空白慢病毒pGCLV-GFP为实验对照组。慢病毒载体经过酶切、连接和包装后进行收集、纯化浓缩、滴度测定及生物安全性检测。
用所获得重组慢病毒pGCLV-sfat1-GFP和慢病毒pGCLV-GFP转化中国西门塔尔牛胎儿成纤维细胞并测定慢病毒最佳MOI,观察并检测牛胎儿成纤维细胞内绿色荧光蛋白(GFP)基因的表达随细胞培养时间变化,提取细胞总RNA进行RT-PCR验证sfat-1基因的表达,使用重组慢病毒感染细胞后对细胞脂肪酸进行提取和成分分析。
实验结果表明:使用密码子替换法和PCR化学合成法能够在体外合成线虫Caenorhabditis.elegans的ω-3脂肪酸脱氢酶基因sfat-1;通过使用携带绿色荧光蛋白基因的慢病毒载体介导线虫sfat-1基因感染中国西门塔尔牛胎儿成纤维细胞能够稳定且高效地表达融合蛋白sfat1-GFP;由sfat-1基因编码的ω-3脂肪酸脱氢酶在牛胎儿成纤维细胞中能够有效地催化多种ω-6 PUFAs转变为相应的ω-3 PUFAs,使二者比例由原来的15.32:1变为0.83:1,并且在牛胎儿成纤维细胞中产生更加丰富的超长链ω-3 PUFAs。
本研究阐述了优化牛胎儿成纤维细胞内脂肪酸成分的有效方法,证明了优化后的线虫ω-3脂肪酸脱氢酶基因sfat-1在动物体细胞内的作用,为在实验和临床研究中制备转基因动物提供了一定的依据。
From epidemiology to cell culture and animal studies to randomized controlled trials, the cardioprotective effects of polyunsaturated fatty acids (PUFAs) are becoming recognized. Recently, Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have been the subject of increasing investigation and have attracted considerable interest as pharmaceutical and nutraceutical compounds. They are subtribe PUFAs and essential components requered for normal cellular function and have been shown to exert many preverntive and therapeutic actions.
An important class of enzymes involved in the synthesis of PUFAs is the fatty acid desaturases, which catalyze the introduction of double bonds into the hydrocarbon chain at a position determined by the enzyme specificity.The Caenorhabditis.briggsae sfat-1 gene, which is absent in most animals, encodes anω-3 fatty acid desaturase that convertsω-6 PUFAs toω-3 PUFAs. The objection of current study was the first attempt to transfer sfat-1 gene with lentivirus vectors in the aim to generate transgenic bovine fetal fibroblasts capable of convertingω-6 PUFAs toω-3 PUFAs.
To heterologously express the n-3 fatty acid desaturase in early bovine fetal fibroblasts, the sfat-1 gene encoding this protein by optimization of codon usage for mammalian cells was modified and was coupled to a pCMV enhancer/chickenβ-actin promoter (which allows high-level and broad expression of the transgenic in all serieses of bovine fibroblasts). The full length sfat-1 gene was constructed by chemical synthesis in vitro by PCR amplification and restriction ligation.
Lentiviral expression plasmid vectors containing GFP domains carrying the sfat-1 gene fragments (pGCLV-sfat1-GFP group) were constructed. Lentivirus with GFP (pGCLV-GFP gourp) domains was performed as a control. After sfat-1 gene fragment transfered, pGCLV-sfat1-GFP group cells and pGCLV-GFP group cells were examined via green fluorescent protein detection. RT-PCR and Western blotting were performed to identify the fusion protein. GC/MS was conducted to analysis the composition of fatty acid by using the extracted total cellular lipids.
Lentivirus-mediate sfat-1 had alterated the composition of fatty acid in Chinese Simmental bovine fetal fibroblasts significantly. Heterologous expression of the sfat-1 gene in bovine fetal fibroblasts capable of converting variousω-6 PUFAs to the correspondingω-3 PUFAs, and changed the n-6/n-3 ratio from about 15.32:1 to 0.83:1.
This study has demonstrated clearly that the sfat-1 gene can be expressed functionally in bovine fetla fibroblasts, and its expression could confer cells’capability of convertingω-6 PUFAs to correspondingω-3 PUFAs, leading to a balanced n-6/n-3 ratio and a change in fatty acids production. The rearch provides an effective approach to modifying fatty acid composition of bovine fetal fibroblasts and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.
引文
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