昆虫学报2012，Vol.55Issue(2)：147-155,9.
华北大黑鳃金龟中肠丝氨酸蛋白酶cDNA克隆、序列分析及表达
Molecular cloning, sequence analysis and expression of serine protease cDNAs from the midgut of Holotrichia oblita (Coleoptera: Melolonthidae)
摘要
Abstract
Serine proteases are one group of important digestive enzymes in insects. To clarify the characteristics and functions of serine proteases, we used the polyclonal antiserum of peritrophic membrane protein from Trichoplusia ni to screen cDNA expression library of the midgut of Holotrichia oblita, and obtained a full-length cDN A clone encoding serine proteases named as HoSPl ( GenBank accession no. FJ573146). The sequence analysis indicated that HoSPl is 902 bp in length with an opening reading frame of 783 bp encoding 260 amino acid residues with the predicted molecular weight 26.7 kDa and pI 4.19. Without N-linked glycosylation site, HoSPl has an O-hnked glycosylation site at Thr157 and six conservative cysteines forming three pairs of disulfide bonds, which play an important role in sustaining the protein tertiary structure. Amino acid sequence alignment with several kinds of serine proteases showed that HoSPl has the catalytic active centers of histidine, aspartic acid and serine, and shares significant similarity to 14 kinds of serine proteases from Costelytra zealandica, with the highest identity (52.47% ) to CzSP3. After the gene was recombined into pET21b and expressed in vitro, the activity of HoSPl was determined with BTEE as the substrate, which was 0. 0378 (junol/mg · min. The molecular cloning and expression in vitro of HoSPl lay a foundation for further research of its expression and function in H. Oblita.关键词
华北大黑鳃金龟/cDNA表达文库/丝氨酸蛋白酶/序列分析/体外表达Key words
Holotrichia oblita-, cDNA expression library/ serine protease/ sequence analysis/ invitro expression分类
生物科学引用本文复制引用
刘海明,郑桂玲,李长友,周洪旭..华北大黑鳃金龟中肠丝氨酸蛋白酶cDNA克隆、序列分析及表达[J].昆虫学报,2012,55(2):147-155,9.基金项目
山东省教育厅项目(J10LC07) （J10LC07）
青岛农业大学博士基金项目(6631024) （6631024）
"泰山学者"建设工程专项经费 （）
