拟蜘蛛拖丝蛋白基因人工合成及其原核表达  

Cloning and Prokaryotic Expression of Artificial Synthetic Spider Dragline Silk Protein Gene

作　　者：王春生[1] 原璐[1] 罗芳[1] 梁洋[1] 安铁洙[1] 

机构地区：[1]东北林业大学生命科学学院,哈尔滨150040

出　　处：《四川动物》2010年第2期184-188,共5页Sichuan Journal of Zoology

基　　金：国家自然科学基金(30751325;30771538)资助;东北林业大学引进人才科研启动基金资助

摘　　要：根据GenBank上蜘蛛拖丝蛋白基因序列(AY555585和AH015065)、拖丝蛋白基因的结构特点和密码子的简并性,设计378bp的拖丝蛋白基因单体,并对其人工聚合成二聚体。Primer5.0分析结果表明,决定拖丝弹性和抗拉力的氨基酸(Gla和Ala)含量(41.43和18.22)接近天然丝蛋白氨基酸含量(42.81和26.32);利用Antheprot软件对二聚体编码氨基酸的二级结构预测结果显示,与丝蛋白的氨基酸链相近,由2个相同的部分排列而成,即β-片层(占48%)、6个α-螺旋间隔(占15%)、散在的转角(占12%)和若干不规则卷曲(占25%)。将二聚体与pET-28a(+)连接构建原核表达载体,IPTG诱导重组菌体裂解物经SDS-PAGE电泳可检测到相对分子质量为26.6×103kD的重组蛋白。上述结果为进一步开展有关转蜘蛛拖丝蛋白基因在绵羊被毛中表达的研究奠定了基础。According to the spider dragline silk protein gene sequence in GenBank （AY555585 and AH015065 ）, the structural characteristics of the gene and degeneracy of codon, a 378 bp sequence was designed, artificially synthesized and duple-polymerized. The amount of Gla and Ala in the synthetic silk. protein gene, which determines elasticity and strength of the synthesized gene, was analogous with that of natural dragline silk protein gene by Primer5. 0 analyzing. Secondary structures of the amino acid sequence was forecasted by Antheprot software, and the results showed that it was composed of the same four segments, including a β-sheet （48%）, α-helix （ 15% ）, dispersed β-turn （ 12% ）, and several anomalistic coil （25%）. These structures accorded with structural characteristics of silk protein, pET-28a（＋）-2S was constructed and expressed in E. coli strain OrigmaiB （DE3）. After IFFG induction, about 26.6 × 10^3 kD recombinant protein was detected in SDS-PAGE. The above results were necessary for further research into spider dragline silk protein gene expression in eukaryotes.

关 键 词：拖丝蛋白基因 人工合成 二聚体 原核表达 

分 类 号：Q78[生物学—分子生物学]