【摘要】：本研究旨在通過(guò)構(gòu)建腺病毒介導(dǎo)的體外超表達(dá)載體,探究腺苷甲硫氨酸轉(zhuǎn)移酶(MAT2B)對(duì)豬肌內(nèi)脂肪細(xì)胞分化的影響。本研究以豬脂肪組織為試驗(yàn)材料,提取總RNA,并反轉(zhuǎn)錄得到cDNA,參考GenBank收錄的豬MAT2B基因mRNA序列設(shè)計(jì)引物,PCR擴(kuò)增并連接到腺病毒穿梭載體pAdTrack-CMV,進(jìn)行測(cè)序鑒定。結(jié)果表明,構(gòu)建的穿梭載體與骨架載體pAdEasy-1可以實(shí)現(xiàn)同源重組,即腺病毒重組載體pAd-MAT2B構(gòu)建成功;用PacⅠ限制性內(nèi)切酶酶切線性化pAd-MAT2B載體并回收質(zhì)粒大片段轉(zhuǎn)染293A細(xì)胞可以實(shí)現(xiàn)病毒的成功包裝,病毒滴度測(cè)定可滿足原代細(xì)胞侵染需要。轉(zhuǎn)染豬原代肌內(nèi)脂肪細(xì)胞后,MAT2B的mRNA和蛋白水平實(shí)現(xiàn)了顯著的上調(diào)。油紅O染色結(jié)果表明,過(guò)表達(dá)MAT2B促進(jìn)了肌內(nèi)脂肪細(xì)胞脂質(zhì)積累;實(shí)時(shí)定量結(jié)果證明,MAT2B促進(jìn)成脂標(biāo)志基因PPARγ和aP2表達(dá)的顯著上調(diào)。綜上所述,腺病毒介導(dǎo)的體外表達(dá)載體可以成功的實(shí)現(xiàn)MAT2B基因超表達(dá);MAT2B正向調(diào)控豬肌內(nèi)脂肪細(xì)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，

本文編號(hào)：2194585