苹果蠹蛾性信息素结合蛋白Ⅱ(CpomPBP2)基因的克隆及原核表达

Cloning and prokaryotic expression of Cydia pomonella pheromone binding proteinⅡ gene(CpomPBP2)

【目的】获得苹果蠹蛾性信息素结合蛋白Ⅱ(CpomPBP2)基因的全长序列,并在大肠杆菌BL21(DE3)中融合表达,为探明苹果蠹蛾雌雄间的信息素交流机制奠定基础。【方法】提取苹果蠹蛾触角总RNA,利用RT-PCR、3′-RACE和5′TAIL-PCR扩增技术获得CpomPBP2基因的全长序列;通过构建原核表达载体pET28aCpomPBP2对CpomPBP2进行重组表达与检测;并对融合蛋白pET28a-CpomPBP2进行Western blot鉴定及可溶性鉴定。【结果】以苹果蠹蛾触角总RNA为模板合成cDNA第一链,以cDNA为模板经RT-PCR、3′-RACE和5′TAILPCR扩增,得到CpomPBP2基因约3 000bp的全长序列;测序结果表明,CpomPBP2基因开放阅读框长约510bp,编码169个氨基酸,预测的成熟蛋白分子质量为16.45ku,等电点(pI)为5.09。经SDS-PAGE电泳分析表明,融合蛋白CpomPBP2以包涵体形式存在,蛋白分子质量约为20ku。Western blot鉴定结果显示,获得了目标蛋白CpomPBP2。用终浓度1.0mmol/L IPTG诱导8h后,可获得大量融合蛋白。【结论】获得CpomPBP2的全长序列,成功构建了CpomPBP2的原核表达载体,经Western blot鉴定,CpomPBP2表达正确。

【Objective】This study obtained the cDNA sequence of pheromone binding protein Ⅱ gene of Cydia pomonella(CpomPBP2)and express CpomPBP2 in Escherichia coli BL21(DE3)to understand the communication mechanism between male and female C.pomonella.【Method】The sequence of CpomPBP2 was obtained using RT-PCR,3′-RACE and 5′TAIL-PCR methods with extracted total RNA fromC.pomonellaantennae.The DNA segment was inserted into expression plasmid pET28a(+)to construct a recombinant expression plasmid and it was expressed and tested.Western blot characterization and soluble form identification of the fused protein pET28a-CpomPBP2 were also conducted.【Result】The fulllength of obtained cDNA was 3 000 bp with a 510 bp open reading frame,which encoded a protein of 169 amino acids with the molecular weight of 16.45 ku and pI value of 5.09.The SDS-PAGE analysis showedthat the cloned fused CpomPBP2 was expressed in the form of inclusion bodies in E.coli BL21(DE3)with a molecular mass of 20 ku.Western blot showed that the fused CpomPBP2 was target protein.Under the1.0mmol/L IPTG and 8hinduction,plenty fused protein was obtained.【Conclusion】The full-length cDNA of CpomPBP2 was obtained and the fused CpomPBP2 was expressed in E.coli BL21(DE3)successfully.Western blot showed that CpomPBP2 was expressed correctly.