家蚕Bm595的表达、组织分布及亚细胞定位

Expression, Tissue Distribution and Subcellular Localization of Bm595 from Silkworm (Bombyx mori)

【目的】从家蚕蛹期cDNA文库中获得一条新基因Bm595,分析Bm595在家蚕体内的表达和定位情况。【方法】利用生物信息学分析Bm595序列,并构建重组表达质粒在大肠杆菌中诱导表达,以纯化的融合蛋白抗原,制备多克隆抗体。用荧光定量PCR和Western blot方法分析Bm595基因在家蚕各组织中的分布。用免疫细胞化学的方法研究Bm595蛋白的定位情况。【结果】Bm595在五龄幼虫的转录水平最高,卵中最低。Bm595在家蚕五龄幼虫各组织中的转录水平由高到低依次为:睾丸、丝腺、肠、头、卵巢、脂肪体、表皮、气管。在表达水平方面,睾丸的表达量最高,其次是丝腺和头,脂肪体有较明显的表达。以家蚕Bm5细胞进行免疫细胞化学试验,Bm595主要分布于细胞核内。【结论】在mRNA、蛋白质表达和细胞水平对Bm595进行初步表达分析和定位分析,为进一步研究该蛋白在家蚕中的功能提供重要依据。

[Objective] In screening the pupa cDNA library, a novel gene Bm595 was identified, the tissue distribution and localization of Bm595 were analyzed in silkworm. [ Method ] The recombinant plasmid was transformed into E.coli BL 21 which were induced with IPTG. The purified fusion protein was used to immune a rabbit and the polyclonal antibody was prepared. The tissue distribution of Bm595 was analyzed by real-time PCR and Western blot. [Result] The quantitative real-time PCR results indicated that the transcription level of Bm595 was the highest in the fifth instar larva and the lowest in egg. The quantity of Bm595 transcription from high to low was testis, silk gland, gut, head, ovary, fat body, epidermis, and air tube, respectively. Western blot results showed that Bm595 was highly expressed in testis, moderately expressed in silk gland and head. Bm595 also existed in fat body. Bm595 was mainly localized in nucleus by immunocytochemistry. [Conclusion] A preliminary analysis on Bm595 was made at the mRNA, protein expression and cell level which laid a good foundation for the further studies on Bm595 function.