摘要
以球毛壳菌cDNA文库中获得过氧化物膜蛋白(pero)基因片段(GenBank Accn: BP099709)为基础,用RACE技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3’ RACE产物和508bp的5’RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17．5kDa,理论等电点为5．75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA 序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明:该基因与粗糙脉孢菌(Neurospora crassa)的过氧化物膜蛋白过敏原同源性最高(83％)。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG 诱导后SDS-PAGE检测表达情况,结果发现在21kDa处有一特异性融合蛋白带,大小与预期相符, 说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在 GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。
The full length cDNA encoding peroxisomal membrane protein from Chaetomium globosum was cloned using RACE technology and the sequence in eDNA library of C. globosum in GenBank （Aeen： BP099709）. The 747bp full length eDNA encoding peroxisomal membrane protein allergen （pero） gene was assembled with 412bp 3＇and 508bp 5＇RACE products. The open reading frame was 501bp encoding 166 amino acids. The molecular weight of the protein was 17.5kDa and its theoretical isoeleetrie point was 5.75. The pero gene was amplified using specific primers of eDNA 5＇and 3＇untranslated region, sequence analysis indicated that the gene have 3 exons and 2 introns. ClustalX analysis revealed that amino acids sequence of pero gene from C. globosum and Neurospora crassa shared 83% high similarity. To construct pET28a-pero expressive plasmid, pero gene was inserted into pET28a expressive vector. Escherichia coli BL21 transformed by pET28a-pero plasmid was induced with IPTG. The protein expression was analyzed with SDS-PAGE. A 21kDa pero fusion protein representing the pero gene was expressed in recombinant E. coli BL21. The sequences of cDNA, DNA and deduced amino acid of the pero gene from C. globosum were submitted to GenBank （Accn： AY555771, AY584753, AAS66898 ）.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2006年第4期40-45,共6页
China Biotechnology
基金
国家"863"计划资助项目(2003AA241140)
关键词
球毛壳菌过氧化物膜蛋白
过敏原基因克隆序列分析原核表达
Chaetomium globosum Peroxisomal membrane Protein allergen Gene cloning Sequence analysis Proeukaryotic expression
