骨骼肌特异表达线虫ω-3脂肪酸去饱和酶基因载体的构建与验证

中国生物工程杂志

2011, Vol. 31

Issue (7): 27-31

研究报告

骨骼肌特异表达线虫ω-3脂肪酸去饱和酶基因载体的构建与验证

于永生^1,2, 张立春², 罗晓彤², 刘铮², 张树敏²

1. 中国农业科技东北创新中心长春 130124;
2. 吉林省农业科学院畜牧分院公主岭 136100

Construction and Verification of Skeletal Muscle Specific Expression Vector of Caenorhabditis Elegans ω-3 Fatty Acid Desaturase Gene

YU Yong-sheng^1,2, ZHANG Li-chun², LUO Xiao-tong², LIU Zheng², ZHANG Shu-min²

1. Northeast Agricultural Research Center of China, Changchun 130124, China;
2. Branch of Animal Husbandry, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China

全文: PDF(544 KB) HTML

摘要：

根据猪α-actin基因已知DNA 序列设计合成了两个特异性引物, 以猪基因组DNA为模板,通过PCR扩增得到α-actin 5'调控序列,然后与线虫ω-3脂肪酸去饱和酶基因cDNA、去除CMV启动子的表达载体pcDNA3.1 连接构成肌肉特异性表达载体pcDNA3.1-AF,小鼠股四头肌注射该重组载体,RT-PCR检测证明其表达有效性。PCR 扩增出1 815 bp的特异性片段, DNA 测序结果证明该克隆片段与α-actin 5'端调控区序列相比具有很高的一致性,含有肌肉特异性元件;小鼠股四头肌注射重组载体,RT-PCR显示肌肉内ω-3脂肪酸去饱和酶基因得到有效转录。

关键词： 猪α -actin; 线虫ω -3脂肪酸去饱和酶; 肌肉特异表达载体; 体外验证

Abstract:

Two specific oligonucleotide primers were designed and synthesized according to the sequence of Sus scrofa skeletal α-actin gene, which were used to amplify the α-actin 5'regulating sequence. pcDNA3.1-AF, the muscle-specific expression vector, was constructed by connecting with α-actin 5'regulating sequence, Caenorhabditis elegans ω-3 fatty acid desaturase cDNA and CMV promoter-less pcDNA3.1,and its expression specificity were proved by directly injecting the vector into the quadriceps muscle of mice(4 weeks old). The PCR amplification result showed that a size of 1 815 base pairs fragment was amplifies and the sequencing resulted showed that the fragment had a high identity with original sequence and contained muscle specific elements. The transient expression results indicated that the muscle-specific expression vector could efficiently direct the transcription of ω-3 fatty acid desaturase in the muscle. The study provides the basis for establishing transgenic animal which could specific express ω-3 fatty acid desaturase in the muscle.

Key words: Sus scrofa skeletal α-actin Caenorhabditis elegans ω-3 fatty acid desaturase Muscle-specific expression vector Verification in vitro

收稿日期: 2011-02-03 出版日期: 2011-07-25

ZTFLH:

Q78

基金资助:

国家自然科学基金资助项目(30970700)

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引用本文:

于永生, 张立春, 罗晓彤, 刘铮, 张树敏. 骨骼肌特异表达线虫ω-3脂肪酸去饱和酶基因载体的构建与验证[J]. 中国生物工程杂志, 2011, 31(7): 27-31.

YU Yong-sheng, ZHANG Li-chun, LUO Xiao-tong, LIU Zheng, ZHANG Shu-min. Construction and Verification of Skeletal Muscle Specific Expression Vector of Caenorhabditis Elegans ω-3 Fatty Acid Desaturase Gene. China Biotechnology, 2011, 31(7): 27-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I7/27

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