家蚕30K蛋白新成员Slp的基因克隆及表达特征与亚细胞定位

Cloning,Expression and Subcelluar Localization of Slp,a New Member of 30K Protein Family from Silkworm,Bombyx mori

家蚕30K蛋白是家蚕生长发育的主要能源物质,具有多种生物学功能。根据已知的30K蛋白的氨基酸序列,利用NCBI在线BLAST程序在家蚕蛋白质数据库中进行氨基酸序列相似性比对分析,发现一条新的氨基酸序列,命名为Slp蛋白(GenBank登录号:NM_001126256)。该蛋白质与已知的5种家蚕30K蛋白的氨基酸序列相似度达41%以上,且具有共同的保守结构域Lipoprotein_11,三级结构也极其相似,推测Slp蛋白可能是家蚕30K蛋白家族新成员。利用NCBI在线工具查找获得了编码Slp的基因序列,然后利用RT-PCR技术从家蚕5龄幼虫脂肪体中克隆得到Slp的cDNA序列,并在大肠杆菌中诱导表达。利用获得的纯化Slp蛋白免疫新西兰大白兔制备多克隆抗体,对该蛋白质进行亚细胞定位的结果显示其主要存在于BmN细胞的细胞膜上和细胞质中。Western blotting检测Slp蛋白在家蚕不同发育时期均有表达,以5龄幼虫期的表达量最高,蛹期次之,蛾期和卵期最低;在5龄幼虫不同组织均有表达,以血淋巴和脂肪体中的表达量最高,在其它组织中的表达量较低。依据Slp蛋白的序列结构与表达特征,推测其与已知的家蚕30K蛋白具有相似的多种生物学功能。

30K proteins are the major energy substance in growth and development of silkworm(Bombyx mori) and possess multiple biological functions.In this study,according to amino acid sequences of the reported 30K proteins,a new silkworm protein named Slp(GenBank accession No.NM_001126256) was found in silkworm protein database from sequence similarity comparison and analysis by using NCBI online BLAST.Its amino acid sequence shares more than 41% identities with other five 30K proteins of silkworm.Moreover,they have the common conserved domain Lipoprotein_11 and highly similar tertiary structure,suggesting that Slp is a new member of 30K protein family in silkworm.The gene sequence encoding Slp was searched and obtained using NCBI online tools.Consequently,cDNA sequence of Slp was cloned from fat body of the 5th instar silkworm larvae by RT-PCR,and was induced to express in E.coli.The purified Slp protein was used to immunize New Zealand rabbit for preparing polyclonal antibodies.Subcellular localization displayed that Slp was distributed mainly in cell membrane and cytoplasm.Western blotting results showed that Slp was widely expressed at different developmental stages of silkworm.The expression level of Slp was the highest at the 5th instar larval stage followed by pupal stage,and was the lowest in adult and egg stages.Meanwhile,Slp was widely expressed in different tissues of the 5th instar larvae.The expression level was the highest in hemolymph and fat body and was relatively lower in other tissues.According to sequence features and expression characteristics,it was presumed that Slp has similar multiple biological functions with other known 30K proteins in silkworm.