家蚕核型多角体病毒遍在蛋白及结合肽的研究  

Study on Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) Ubiquitin and Its Binding Peptides

作　　者：张文波[1,2] 林蓉[3] 张耀洲[4] 吴祥甫[5] 

机构地区：[1]浙江大学生命科学学院 [2]浙江理工大学生物化学研究所,杭州310018 [3]浙江大学动物科学学院 [4]浙江理工大学生物化学研究所 [5]中国科学院生物化学与细胞生物学研究所

出　　处：《生物化学与生物物理进展》2004年第11期1038-1044,共7页Progress In Biochemistry and Biophysics

摘　　要：从家蚕核型多角体病毒镇江株 (BombyxmorinuclearpolyhedrosisvirusZhenjiangstrain ,BmNPV ZJ)基因组DNA中克隆遍在蛋白 (BmVUB)基因 .序列分析结果显示 ,BmVUB基因长 2 34bp ,编码 77个氨基酸 .BmVUB氨基酸序列内有一致HTH序列 ,遍在蛋白保守序列LRLRGG ,参与遍在蛋白 蛋白酶复合体形成的 4个保守性功能位点 (Lys 2 9、Cys 4 8、Cys 6 3、Gly 76 )及保守的Gly Gly X (X是疏水氨基酸残基 )蛋白酶切信号序列 ,在遍在蛋白保守的Gly Gly后多出由一个氨基酸组成的延伸肽 .原核诱导表达表明BmVUB主要以可溶性形式存在 ,表达量占总菌蛋白的 5 0 %以上 .纯化的遍在蛋白浓度为 1 0 3～ 2 4 6 g/L ,制备抗体 ,效价在 3 2× 10 -5以上 .用噬菌体表面展示技术筛选遍在蛋白结合肽 ,所筛选的结合肽可与遍在蛋白特异性结合 ,其中结合肽VAPHHAYAPMRT对细胞增殖有明显的浓度调控作用 ,即低浓度促进细胞生长 ,高浓度强烈抑制细胞的生长 .The ubiquitin (UB) gene of Bombyx mori nuclear polyhedrosis virus Zhenjiang strain (BmNPV-ZJ) was cloned and sequenced. Sequencing analysis shows that the BmVUB gene, which contains 234 nucleotides encoding 77 amino acids, contains a conservative HTH, a conservative LRLRGG and four conservative functional sites: Lys-29, Cys-48, Cys-63, Gly-76 which play important roles in ubiquitin-proteasome complex formation. It also shows that the C-terminal amino acid was redundant which locate at the downstream of conservative Gly-Gly proteinase cleavage signal sequence which characterizes as Gly-Gly-X ( X was hydrophobic amino acid). The BmVUB gene was induced and highly expressed in E. colt BL21. The results of gel scanning and Bradford protein assay show that the BmVUB protein was about 9 ku and the yield consists of 50 percent of total bacterial protein. The concentration of purified BmVUB protein was 1.03 similar to 2.46 g/L. The high titer antibody wag obtained from rabbits immunized with the (His)(6) - BmVUB fusion protein and ELISA shows that the titer of antibody was over 3.2 X 10(-5). Peptides binding ubiquitin were obtained from phage displayed peptide library using BmNPV-ZJ ubiquitin purified with His-affinity chromatography as a ligand. Five short peptides were identified which displayed obvious biological activity. Peptide VAPHHAYAPMRT could regulate the cell proliferation and further more this function which depends on the concentration variation, namely low concentration could promote the cell proliferation, whereas high concentration will inhibit the proliferation.

关 键 词：结合肽 遍在蛋白 白及 性功能 原核 细胞增殖 细胞生长 诱导表达 氨基酸序列 特异性结合 

分 类 号：Q78[生物学—分子生物学] R742.5[医药卫生—神经病学与精神病学]