【摘要】 脂围蛋白(perilipin)与脂肪细胞脂类代谢的调节有关。从家蚕蛹cDNA文库中筛选到一条cDNA开放阅读框(ORF)序列长度为918 bp的基因,预测其编码305个氨基酸残基,蛋白分子质量约32.5 kD。经NCBI比对发现该蛋白具有部分脂围蛋白的保守结构域,因此将该ORF序列命名为BmPLIN基因。将该基因插入到载体pET-28a(+)的多克隆位点,构建重组表达质粒pET-28a(+)-BmPLIN,并转化大肠杆菌中进行原核表达得到带有His标签的融合蛋白,表达的融合蛋白以包涵体形式存在。用纯化后的融合蛋白为抗原免疫大白兔获得多克隆抗体,ELISA间接法测定抗体的效价达到1∶12 800以上。通过荧光定量PCR和Western blotting技术对BmPLIN基因及其编码蛋白在家蚕5龄幼虫各个组织器官中的转录、表达水平进行分析的结果是:BmPLIN基因mRNA在家蚕5龄幼虫各组织器官中的转录水平由高到低依次为生殖腺(卵巢和睾丸)、脂肪体、肠、表皮、头部、马氏管、丝腺、气门,在家蚕5龄幼虫的生殖腺(卵巢和睾丸)中有明显的BmPLIN蛋白特异条带。以家蚕Bm5细胞进行免疫细胞实验的结果显示,BmPLIN蛋白在细胞质和细胞核中均有分布。更多还原

【Abstract】 Perilipin protein is involved in lipid metabolism in adipocytes.A cDNA with 918 bp open reading frame(ORF) was obtained through screening the previously constructed cDNA library for pupal stage of the silkworm,Bombyx mori.The ORF encodes a perilipin protein of 305 amino acid residues with a predicted molecular weight of 32.5 kD.Sequence alignment on NCBI website revealed that the protein has a conservative structural domain of perilipin and was thus named BmPLIN.After the ORF was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli Rosetta,a fusion protein with His tag was successfully expressed as inactive inclusion bodies.After purification,the fusion protein was used as antigen to obtain polyclonal antibodies from rabbit.The titer of the polyclonal antibodies measured by ELISA was beyond 1∶12 800.The transcription and expression levels of BmPLIN gene in different tissues of the fifth instar larvae of Bombyx mori were studied using fluorescence quantitative PCR and Western blotting etc.The results indicated that the transcriptional level of BmPLN mRNA in different tissues of the fifth instar larvae was in the order of gonad > fat body > gut > epidermis > head > Malpighian tubule > silk gland > spiracle,and the gonad of the fifth instar larvae had obvious BmPLIN protein specific band.Moreover,subcellular localization experiment with Bm5 cells displayed that BmPLIN protein existed in both cytoplasm and nucleus.更多还原