【摘要】：試驗旨在克隆從江香豬載脂蛋白A1(apolipoprotein A1,ApoA1)基因,研究ApoA1基因在真核細(xì)胞中的亞細(xì)胞定位情況。通過提取從江香豬總RNA,采用RT-PCR、目的基因的連接、轉(zhuǎn)化等方法構(gòu)建攜帶有綠色熒光蛋白的pEGFP-C1-ApoA1重組質(zhì)粒,并經(jīng)菌落PCR、雙酶切及測序鑒定正確后,轉(zhuǎn)染HEK-293T細(xì)胞,36h后觀察熒光,分析ApoA1蛋白在真核細(xì)胞中的亞細(xì)胞定位情況。結(jié)果表明,從江香豬ApoA1基因與GenBank上公布的野豬序列相比,有6處發(fā)生了堿基突變,其中5處為有義突變,分別導(dǎo)致180位氨基酸由丙氨酸變?yōu)楣劝彼帷?85位氨基酸由組氨酸變?yōu)楣劝滨０贰?86位氨基酸由纈氨酸變?yōu)榱涟滨０贰?09位氨基酸由天冬氨酸變?yōu)楦拾彼?PSORTⅡPrediction和熒光共定位試驗結(jié)果均表明,ApoA1蛋白的表達主要集中在細(xì)胞外基質(zhì),約占總表達量的77.8%。本試驗成功克隆了從江香豬ApoA1基因CDS區(qū),且ApoA1蛋白的表達主要集中在細(xì)胞外基質(zhì)中,為進一步構(gòu)建ApoA1基因轉(zhuǎn)基因動物模型、開展ApoA1基因與人類因肥胖引起的相關(guān)疾病關(guān)系的研究奠定基礎(chǔ)。
[Abstract]:The aim of this study was to clone the apolipoprotein A1 (apolipoprotein A1) gene of Congjiang Xiang pig and to study the subcellular localization of ApoA1 gene in eukaryotic cells. The recombinant plasmid carrying green fluorescent protein (GFP) was constructed by extracting total RNA, from Congjiang Xiang pig by ligation and transformation of RT-PCR, target gene. The recombinant plasmid was confirmed by double digestion of colony PCR, and sequencing. After transfection of HEK-293T cells, fluorescence was observed after 36 h, and the localization of ApoA1 protein in eukaryotic cells was analyzed. The results showed that there were 6 base mutations in the ApoA1 gene of Congjiang Xiang pig compared with the wild boar sequence published on GenBank, 5 of which were semantic mutations, which led to the amino acid change from alanine to glutamate, respectively. 185 amino acids changed from histidine to glutamine, 186 amino acids changed from valine to leucine, and 209 amino acids changed from aspartic acid to glycine. The results of PSORT 鈪，

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