| 摘要: |
| 【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pET28a-CpomPBP2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′ TAI PCR扩增，得到CpomPBP2基因约3 000 bp的全长序列；测序结果表明，CpomPBP2基因开放阅读框长约510 bp，编码169个氨基酸，预测的成熟蛋白分子质量为16.45 ku，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20 ku。Western blot鉴定结果显示，获得了目标蛋白CpomPBP2。用终浓度1.0 mmol/L IPTG诱导8 h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。 |
| 关键词: 苹果蠹蛾 苹果蠹蛾性信息素结合蛋白Ⅱ基因 基因克隆 原核表达 |
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| 基金项目:公益性行业(农业)科研专项(200903042-03)；陕西省“13115”重大科技专项(2009ZDKG-06) |
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| Cloning and prokaryotic expression of Cydia pomonella pheromone binding protein Ⅱ gene (CpomPBP2) |
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CAO Xin-yue,HUANG Ai-yuan,ZHAO Xiao,et al
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| Abstract: |
| 【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 from C.pomonella antennae.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 full-length 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 showed that 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 the 1.0 mmol/L IPTG and 8 h induction,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. |
| Key words: Cydia pomonella pheromone binding protein Ⅱ gene of Cydia pomonella(CpomPBP2) gene cloning prokaryotic expression |
