腺病毒介导的MAT2B过表达促进猪肌内脂肪细胞分化

摘要

The aim of this study was to explore the effect of MAT2B on porcine intramuscular preadipocyte differentiation by constructing overexpression vector in vitro mediated by adenovirus.A pair of exclusive primers was designed according to the GenBank sequence information of MAT2B gene,and MAT2B was amplified by PCR.The obtained PCR products were inserted into a shuttle vector pAdTrack-CMV,then the gene was identified by sequencing.The results showed that shuttle vector (pAdTrack-CMV) and backbone vector (pAdEasy-1) were in homologous recombination,and the adenovirus recombinant vector (pAd-MAT2B) was constructed successfully.Then the recombinant adenovirus DNA was digested by the Pac I,the pAd-MAT2B adenovirus was produced,and we used it to infect the porcine preadipocytes.The expression level of mRNA and protein of MAT2B significantly increased.Oil Red O staining assay showed that overexpression of MAT2B promoted lipid accumulation in intramuscular preadipocyte.Moreover,the expression of adipogenic gene PPARγ and aP2 mRNA were significantly promoted when infected by Ad-MAT2B.The results suggest that MAT2B is overexpressed in expression vector in vitro mediated by adenovirus,and MAT2B play a positive role during porcine intramuscular preadipocyte differentiation.%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MA T2B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Pac Ⅰ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,MA T2B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MA T2B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.