腺病毒介导的MAT2B过表达促进猪肌内脂肪细胞分化
发布时间：2018-08-20 18:26
【摘要】：本研究旨在通过构建腺病毒介导的体外超表达载体,探究腺苷甲硫氨酸转移酶(MAT2B)对猪肌内脂肪细胞分化的影响。本研究以猪脂肪组织为试验材料,提取总RNA,并反转录得到cDNA,参考GenBank收录的猪MAT2B基因mRNA序列设计引物,PCR扩增并连接到腺病毒穿梭载体pAdTrack-CMV,进行测序鉴定。结果表明,构建的穿梭载体与骨架载体pAdEasy-1可以实现同源重组,即腺病毒重组载体pAd-MAT2B构建成功;用PacⅠ限制性内切酶酶切线性化pAd-MAT2B载体并回收质粒大片段转染293A细胞可以实现病毒的成功包装,病毒滴度测定可满足原代细胞侵染需要。转染猪原代肌内脂肪细胞后,MAT2B的mRNA和蛋白水平实现了显著的上调。油红O染色结果表明,过表达MAT2B促进了肌内脂肪细胞脂质积累;实时定量结果证明,MAT2B促进成脂标志基因PPARγ和aP2表达的显著上调。综上所述,腺病毒介导的体外表达载体可以成功的实现MAT2B基因超表达;MAT2B正向调控猪肌内脂肪细胞分化。
[Abstract]:The aim of this study was to investigate the effect of adenosine methionine transferase (MAT2B) on the differentiation of porcine intramuscular adipocytes by construction of adenovirus mediated overexpression vector in vitro. In this study, total RNAs were extracted from porcine adipose tissue, and cDNAs were obtained by reverse transcription. The recombinant plasmid pAdTrack-CMV was amplified and ligated to adenovirus shuttle vector pAdTrack-CMV by PCR with reference to the mRNA sequence of porcine MAT2B gene included in GenBank. The results showed that the constructed shuttle vector and skeleton vector pAdEasy-1 could realize homologous recombination, that is, adenovirus recombinant vector pAd-MAT2B was successfully constructed. The successful packaging of 293A cells could be realized by using Pac 鈪，
本文编号：2194585
[Abstract]:The aim of this study was to investigate the effect of adenosine methionine transferase (MAT2B) on the differentiation of porcine intramuscular adipocytes by construction of adenovirus mediated overexpression vector in vitro. In this study, total RNAs were extracted from porcine adipose tissue, and cDNAs were obtained by reverse transcription. The recombinant plasmid pAdTrack-CMV was amplified and ligated to adenovirus shuttle vector pAdTrack-CMV by PCR with reference to the mRNA sequence of porcine MAT2B gene included in GenBank. The results showed that the constructed shuttle vector and skeleton vector pAdEasy-1 could realize homologous recombination, that is, adenovirus recombinant vector pAd-MAT2B was successfully constructed. The successful packaging of 293A cells could be realized by using Pac 鈪，
本文编号：2194585
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