家蚕BmLHep_59蛋白的表达和亚细胞定位

Expression and Subcellular Localization of Bombyx mori BmLHep_59 Protein

肝细胞癌相关抗原-59(hepatocellular carcinoma-associated antigen59,Hep_59)属于哺乳动物中一类含有约100个氨基酸残基的保守结构域家族,这类蛋白主要存在于真核生物细胞中,大多为假想蛋白。从家蚕蛹cDNA文库中筛选的一个新基因,其编码蛋白含有一个和Hep_59高度同源的保守结构域,将其命名为BmLHep_59(GenBank登录号:DN985181)。PCR扩增该基因的ORF,经EcoRⅠ/XhoⅠ双酶切重组到原核表达载体pET-28a(+),并转入E.coli Rosetta(DE3)感受态细胞中表达。通过镍柱亲和层析得到纯化的重组蛋白BmLHep_59,并以此纯化重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体,经ELISA检测抗血清效价可达1∶12 000以上。用Western blotting检测到家蚕5龄幼虫的表皮、丝腺和血淋巴中均有BmLHep_59表达,其中在血淋巴的表达量较高,暗示该蛋白可能参与调控家蚕的造血机能。该蛋白在蛹期中也有表达。亚细胞定位显示BmLHep_59在家蚕Bm5细胞的细胞质和细胞核中均有分布。

Hepatocellular carcinoma-associated antigen 59（Hep_59） is one member of the conserved domain family consisting of 100 amino acid residues found mainly in mammals.These proteins mainly exist in eukaryotic cells and most of them are hypothetical proteins.A new gene was obtained from screening the pupa cDNA library of silkworm（Bombyx mori）.Its encoded protein had a conserved domain homologous to Hep_59 and was thus designated as BmLHep_59（GenBank accession number DN985181）.Open reading frame（ORF） of the gene was amplified by PCR and cloned into the prokaryotic expression vector pET-28a（＋） through EcoRⅠ/XhoⅠdual enzyme digestion.The recombinant plasmid was transformed into competent E.coli Rosetta（DE3） cells.After being purified with Ni-NTA chromatography,the recombinant protein BmLHep_59 was used as antigen to immunize New Zealand rabbit for preparing polyclonal antibody.ELISA analysis showed that titer of the antiserum was over 1 ∶12 000.Western blotting analysis indicated that BmLHep_59 was expressed in epidermis,silk gland,and hemolymph of the 5th instar silkworm larvae,among which the highest expression was seen in hemolymph,suggesting its involve-ment in the regulation of hematopoiesis in silkworm.This protein was also expressed at pupa stage.Subcellular localization showed that BmLHep_59 distributed in both cytoplasm and nucleus of silkworm Bm5 cells.