家蚕BmeIF3s5基因的克隆、表达及亚细胞定位研究
首发时间:2009-12-09
摘要:真核生物翻译起始因子(eIFs)在蛋白合成中起关键作用,在已经鉴定的13个因子中elF3(由8个或更多的亚基组成)是分子量最大的一个并在翻译起始过程中起着核心作用。eIF3s5是eIF3的其中一个亚基并介导了eIF3大多数功能的实现。我们通过RT-PCR的方法从家蚕蛹中扩增到家蚕eIF3s5基因的完整的ORF序列,并将其命名为BmeIF3s5(Bombyx mori eIF3s5),GenBank登录号为DQ443287,该基因序列长为834bp,编码278个氨基酸,预测分子量为29.60kD。我们把BmeIF3sf基因克隆到pET-28a(+)载体上,并在大肠杆菌中成功表达出重组融合蛋白;纯化融合蛋白并免疫新西兰雄性大白兔获得多克隆抗体血清,ELISA检测抗体效价达到1:51200以上。用免疫印迹法实验分析BmeIF3s5蛋白在家蚕不同时期和不同组织中的表达情况,结果表明BmeIF3s5在所检测家蚕的各发育时期和各组织中均有不同程度的表达,其中丝腺、卵、蛾的表达量较高。通过亚细胞定位发现该蛋白主要存在于细胞质中。本研究为进一步探讨BmeIF3s5蛋白在家蚕生长、发育过程中的生物学功能奠定了基础。
关键词: 家蚕 真核翻译起始因子3亚基5 免疫印迹法 亚细胞定位
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The Cloning, expression and subcelluar localization analysis of the BmeIF3s5 gene from silkworm (Bombyx mori)
Abstract:Eukaryoctic initiation factors (eIF3s) are the key elements in protein synthesis.Among identified 13 initiation factors, eIF3,composed of 8 or more subunits, is the largest one and plays a central role in the initiation of translation.eIF3s5 is one subunits of eIF3 complex and mediates most functions of eIF3. The full open reading frame (ORF) of Bombyx mori eIF3s5 was obtained from silkworm pupa by RT-PCR and named BmeIF3s5, of which the accession number was DQ443287. This ORF contained 834 bp, encoding a polypeptide of 278 amino acids with a predicted molecular weight of 29.60 kDa.We cloned the BmeIF3s5 gene and inserted it into pET-28a(+) vector, then induced the expression of recombinant protein in E.coli.In order to obtain polyclonal antibody, the purified fusion protein was used to immunize a male New Zealand rabbit. The titer of the ployclonal antibody reaches 1:51200 measured by ELISA.Western blotting validated the antibody was specific to His-BmeIF3s5. The total proteins from eight developmental stages and ten different tissues of silkworm were extracted for Western blotting, in order to analysis the expression level of BmeIF3s5. Our results showed that BmeIF3s5 was existed in all development stages and organizations test at different levels of expression,especially higher in silk gland,egg and adult.The experiment with ploy-antibody indicated that BmeIF3s5 was mainly distributed in cytoplasm through the method of subcellular localization.It laid a good foundation for further researching the biological function of BmeIF3s5 gene,which was in the growth and development process of silkworm.
Keywords: Bombyx mori Eukaryoctic initiation factor 3 subunit 5 Western blotting Subcelluar localization
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