家蚕类胰岛素相关肽结合蛋白2(BmIBP2)的原核表达与组织特异性分析

Prokaryotic Expression of Bombyx mori Insulin-Related Peptide-Binding Protein 2 (BmIBP2) and Its Tissue-Specificity in Silkworm

【目的】从体外表达家蚕中新发现的1个免疫球蛋白超家族成员—类胰岛素相关肽结合蛋白2(BmIBP2),并制备其多克隆抗体,为进一步深入研究BmIBP2的生物学功能奠定基础。【方法】以家蚕中肠的总cDNA为模板,利用普通PCR方法,扩增出BmIBP2基因的成熟肽序列;通过构建重组表达质粒pET-28a-BmIBP2对BmIBP2的成熟肽进行重组表达;制备多克隆抗体,通过免疫共沉淀的方法研究BmIBP2蛋白的组织特异性。【结果】SDS-PAGE电泳分析表达产物发现,重组蛋白rBmIBP2主要以包涵体的形式表达,目的蛋白大小约为30 kD;以兔抗His-tag抗体为一抗进行Western blot检测,结果表明rBmIBP2具有良好的免疫反应性;用镍离子亲和层析的方法可获得纯化的rBmIBP2,以纯化的rBmIBP2为抗原免疫新西兰白兔制备多克隆抗体,Western blot结果显示家蚕的中肠组织匀浆液中有1条约28 kD大小的条带,大小与BmIBP2成熟肽的蛋白分子量基本一致,而脂肪体、血淋巴、丝腺、精巢及卵巢的组织匀浆液中没有检测到特异反应条带。【结论】成功地实现了BmIBP2在大肠杆菌中的表达和纯化,并获得其多克隆抗体,Western blot结果证明BmIBP2被正确表达,并明确了该蛋白只在家蚕中肠表达的组织特异性。

[Objective] The objective of this study is to express insulin-related peptide-binding protein 2 from silkworm （BmIBP2） which belongs to the immunoglobulin （Ig） superfamily in vitro and prepare polyclonal antibody against recombinant BmIBP2 for the further research about its biological function. [Method] The gene fragment of the mature BmIBP2 peptide was amplified by PCR method with the eDNA synthesized from silkworm midgut. In order to express the protein of mature BmIBP2 peptide, the DNA segment was inserted into the expression plasmid pET-28a（＋） to construct a recombinant expression plasmid. Polyclonal antibody was prepared to study the tissue-specificity expression of BmIBP2 protein in silkworm midgut. [Result] The SDS-PAGE result showed that the cloned recombinant BmIBP2 （rBmIBP2） was expressed in the form of inclusion bodies in E. coli BL21 with a molecular weight of 30 kD, and Western blot using the antibody against His-tag as the primary antibody indicated thatrBmlBP2 had satisfied immunobiological activity. Pure protein was obtained by Ni2＋ NTA resin column to prepare the anti-rBmlBP2 polyclonal antibody by immunizing New Zealand white rabbit. Western blot analysis showed that BmlBP2 protein could be bound with the polyclonal antibody in silkworm midgut with a molecular weight of 28 kD, which is consistent with the predicted molecular of BmIBP2 mature peptide. However, no specific bands were found in other tissues including fat body, hemocytes, silk gland, testicle and ovary. [Conclusion] Recombinant BmIBP2 protein was successfully expressed in E. coli BL21, and its polyelonal antibody was prepared in this study. Moreover, Western blot showed that BmlBP2 was correctly expressed in vitro and had a tissue-specificity expression in silkworm midgut.