中国农业科学 ›› 2008, Vol. 41 ›› Issue (3): 933-938 .doi: 10.3864/j.issn.0578-1752.2008.03.041

• 研究简报 • 上一篇    

中华蜜蜂气味结合蛋白ASP2 cDNA的克隆及原核表达

李红亮,聂文敏,高其康,程家安   

  1. 中国计量学院生命科学学院
  • 收稿日期:2006-08-09 修回日期:1900-01-01 出版日期:2008-03-10 发布日期:2008-03-10
  • 通讯作者: 高其康

Cloning of cDNA encoding Odorant Binding Protein ASP2 in Working Bee’s Antenna of Apis cerana cerana and its Prokaryotic Expression

  

  1. 中国计量学院生命科学学院
  • Received:2006-08-09 Revised:1900-01-01 Online:2008-03-10 Published:2008-03-10

摘要: 【目的】克隆、分析和原核表达编码中华蜜蜂气味结合蛋白ASP2的cDNA。【方法】以13个不同日龄中华蜜蜂工蜂触角为材料,通过RT-PCR技术以获得编码中华蜜蜂Apis cerana cerana气味结合蛋白ASP2基因成熟蛋白开放阅读框序列,并在pET-30a(+)/BL21(DE3)系统中进行原核表达。【结果】克隆了命名为Acer-ASP2(GenBank登录号:DQ449667)的中华蜜蜂气味结合蛋白ASP2基因,该序列全长429 bp,编码142个氨基酸残基,预测分子量和等电点分别为15.7 kD和4.36。预测蛋白具有昆虫气味结合蛋白典型的6个保守的半胱氨酸残基的特征,进一步分析表明Acer-ASP2极有可能属于GOBP家族。构建的原核表达载体pET/Acer-ASP2,在大肠杆菌BL21(DE3)中成功地表达出一个分子量约为21kD的融合蛋白,融合蛋白大部分以包涵体的形式存在于菌体沉淀中(约59.7%),经洗涤和尿素梯度溶解对包涵体进行了纯化,经凝胶光密度扫描分析,约占最终产物的81.2%左右。【结论】克隆、分析和表达了一个新的编码中华蜜蜂气味结合蛋白ASP2 cDNA序列,为进一步研究其分子结构和功能奠定了基础。

关键词: 中华蜜蜂, 工蜂触角, 气味结合蛋白, 原核表达

Abstract: 【OBJECTIVE】Cloning and expression in prokaryotic system of a new cDNA, named Acer-ASP2, encoding odorant binding protein ASP2(antenna special protein 2) open reading frame(ORF).【METHOD】Based on the antenna materials of 13 ages in days from Apis cerana cerana, reverse transcription-polymerase chain reaction(RT-PCR) and pET-30a(+)/BL21(DE3) prokaryotic expression system, Acer-ASP2 was cloned and expressed. 【RESULTS】The full length of Acer-ASP2(GenBank loucs: DQ449667) was 429bp, encoding 142 amino acids and the predicted MW and pI were 15.7 kD and 4.36, respectively. Sequencing and analysis indicated that Acer-ASP2 was characterized by six conservative Cys, which shared typical feature of OBP from other insects. Further analysis showed Acer-ASP2 likely belongs to the family of GOBP. The molecular weight of recombinant protein of pET/Acer-ASP2 was about 21kD, induced by IPTG to highly express a protein of as an inclusion body and accumulated up to 59.7% of bacterial total protein.【CONCLUSION】We have cloned and expressed a new cDNA of Apis cerana cerana, for the further research to the molecular structure and function.

Key words: Apis cerana cerana, Working bee´, s antenna, Odorant Binding Protein, Prokaryotic express