中华蜜蜂气味结合蛋白ASP2 cDNA的克隆及原核表达

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

【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429bp,编码142个氨基酸残基,预测分子量和等电点分别为15.7kD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ASP2cDNA序列,为进一步研究其分子结构和功能奠定了基础。

【Objective】Cloning and expression in prokaryotic system of a new cDNA, named Acer-ASP2, encoding odorant binding protein ASP2 （antenna special protein 2） from Apis cerana cerana.【Method】Based on the antenna materials of 13 working bees of different 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. 【Result】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 with other insects. Further analysis showed that Acer-ASP2 may belong to the family of GOBP. The molecular weight of recombinant protein of pET/Acer-ASP2 was about 21 kD, induced by IPTG to highly express a protein as an inclusion body. The purified inclusion body by washing and dissolving of urea accumulated up to 81.2% of the final products.【Conclusion】We have cloned and expressed a new cDNA of Apis cerana cerana, is closed and expressed, thus laying foundation for further research on molecular structure and function.