家蚕一种富含半胱氨酸蛋白基因BmCRP的原核表达与定位研究

从家蚕蛹cDNA文库中获得一条cDNA序列,经生物信息学分析显示该cDNA序列全长1 011 bp,包括5′-UTR 200 bp和3-′UTR 136 bp,编码224个氨基酸,其编码蛋白质的N-端富含半胱氨酸,将该基因命名为BmCRP(基因登录号:DN985186.1)。该基因编码蛋白主要有α螺旋、β-折叠、无规则卷曲3种二级结构,具有cAMP和cGMP依赖的蛋白激酶磷酸化位点等5种功能位点。构建重组表达载体pET-28 a(+)-BmCRP并转化至大肠杆菌BL21(DE3),经IPTG诱导后获得融合蛋白,用纯化后的目的蛋白免疫新西兰大白兔制备的多克隆抗体与BmCRP有较好的特异性。W estern b lotting检测结果:BmCRP蛋白在家蚕卵、幼虫、蛹、蛾4个发育时期均有表达,蛹期表达量最高;在5龄幼虫的表皮、头部、卵巢、睾丸、马氏管中均有表达。荧光定量RT-PCR检测结果:BmCRPmRNA在家蚕卵、幼虫、蛹、蛾的整个生命周期中,其转录水平呈增加趋势;在5龄幼虫不同组织中的转录水平从高到低依次为表皮、头部、生殖腺、中肠、马氏管、气管、丝腺和脂肪体。细胞定位实验结果显示BmCRP蛋白在Bm5细胞的细胞核和细胞质中均存在,细胞核中的荧光信号比细胞质中的信号强。研究结果有助于进一步探明家蚕BmCRP蛋白的结构和功能。

Study on Prokaryotic Expression and Localization of a Cysteine-rich Protein Gene(BmCRP) in Bombyx mori

An unique new Bombyx mori cDNA was identified from the pupal cDNA library constructed in our laboratory.Sequence analysis revealed that the full-length cDNA is 1 011 bp long with 200 bp 5′-UTR and 136 bp 3′-UTR.This gene codes for a protein with 224 amino acids which is rich in cysteine at the N-terminus and was thus named BmCRP(Bombyx mori cysteine-rich protein) gene(GenBank accession number DN985186.1).This protein has such secondary structures as α-helix,β-pleated sheet and random coil and possesses 5 functional sites including cAMP-and cGMP-dependent protein kinase phosphorylation site.The ORF of the gene was cloned into pET-28a(+) vector and transformed into E.coli BL21(DE3).Recombinant protein was expressed successfully in E.coli BL21(DE3) induced by IPTG with the final concentration of 1 mM.Polyclonal antibodies were generated by immunizing New Zealand rabbit with purified recombinant protein.Western blotting analysis showed that the antibody could bind His-BmCRP specifically.Expression of the protein was observed in various developmental stages including egg,larva,pupa and moth,among which pupa had the highest expression level.The expression was also observed in tissues such as epidermis,brain,ovary,testes and Malpighian tubule of the fifth instar larvae.Real-time quantitative PCR results indicated that the expression level of BmCRP mRNA in silkworm egg,larva,pupa and moth increased gradually;and that in epidermis,brain,ovary,testis,midgut,Malpighian tubule,trachea,silk gland and fat body of the fifth instar larvae decreased gradually.Immunocytochemistry in Bm5 cells showed that BmCRP existed in the nucleus and cytoplasm.The fluorescent signal from nucleus was stronger than that from cytoplasm.These results laid a good foundation for further study on BmCRP′s structure and function in silkworm.