腺病毒介导的MAT2B过表达促进猪肌内脂肪细胞分化

MAT2B Overexprssion Mediated by Adenovirus Promoting 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。

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.