淡足侧沟茧蜂气味结合蛋白MpOBP8的基因克隆及原核表达分析

Molecular cloning and prokaryotic expression of the odorant binding protein MpOBP8 in Microplitis pallidipes Szepligeti

【目的】MpOBP8是淡足侧沟茧蜂Microplitis pallidipes Szepligeti的气味结合蛋白,对于昆虫感知外界气味物质起着不可替代的作用。本研究旨在通过MpOBP8的克隆及其优化表达,获得可溶性目的蛋白,探索其嗅觉识别的分子机理。【方法】通过半定量PCR对其组织差异表达进行分析;通过双酶切构建融合表达载体pET32a(+)-MpOBP8,在大肠杆菌中对其进行原核表达分析并对其表达条件进行了优化。【结果】克隆得到了淡足侧沟茧蜂MpOBP8基因(GenBank登录号:MG457156),该基因含有459 bp的开放阅读框,编码152个氨基酸,推测分子量为17.57ku,预测PI值为4.78。通过半定量PCR,在触角中检测到MpOBP8的表达,而在头、胸、腹中均未检测出表达。对其原核表达条件的优化显示:最佳诱导温度、诱导时间、诱导时机、异丙基-β-D-硫代半乳糖苷(IPTG)的终浓度分别为25℃、6 h、菌体培养液OD_(600)为0.1-0.3和0.1 mmol·L^(-1)。大多数的靶蛋白能以上清形式表达,并通过纯化和浓缩得到了高浓度的靶蛋白。【结论】该研究克隆了淡足侧沟茧蜂的MpOBP8基因,并对其原核表达条件进行了优化,探明了该蛋白的最优表达条件,大量获得该气味结合蛋白,为后期研究MpOBP8的嗅觉识别分子机理奠定基础。

[Objectives]MpOBP8 is an odorant binding protein(OBP)of Microplitis pallidipes,which plays an essential role in the perception of external odors.The aim of this study was to clone the OBP,optimize its expression,and explore the molecular mechanism of olfactory recognition.[Methods]Differential expression in different tissues was analyzed with semi-quantitative PCR.A fusion expression vector pET32a(+)-MpOBP8 was constructed using double enzyme digestion.The plasmid pET32a(+)-MpOBP8 was transferred into the Escherichia coli and the expression conditions were optimized.[Results]The MpOBP8 gene(GenBank accession number:MG457156)was cloned.The open reading frame was 459 bp,which encoded a polypeptide of 152 amino acids.The protein molecular mass was predicted as 17.57 ku with an isoelectric point of 4.78.The expression of MpOBP8 was detected in the antennae with semi-quantitative PCR,but no expression was detected in the head,thorax or abdomen.The best induction temperature,induction time,and the final IPTG concentration were 25℃,6 h and 0.1 mmol·L-1,respectively,whereas the induction opportunity for protein expression at culture medium with OD600 was up to 0.1-0.3.We obtained most target protein under the above conditions and a high concentration by purification and concentration.[Conclusion]We successfully cloned the MpOBP8 gene and optimized the conditions for its prokaryotic expression,obtaining a large amount of the odorant binding protein.These results provide a potential basis for exploring the molecular mechanism underlying olfactory recognition.