管氏肿腿蜂毒液过敏原3基因的克隆及表达分析

Cloning and expression analysis of venom allergen 3 gene of Scleroderma guani

为研究管氏肿腿蜂Scleroderma guani毒液过敏原3基因SgA3的功能,通过RT-PCR克隆SgA3基因,采用荧光定量PCR分析其在不同发育阶段和组织中的表达特征,并利用载体pSUMO-Mut对其进行原核表达。克隆获得SgA3基因开放阅读框长699 bp,编码233个氨基酸,其中信号肽位于N端第1~23位氨基酸。多序列比对分析发现,SgA3与丽蝇蛹集金小蜂Nasonia vitripennis、粉蝶盘绒茧蜂Cotesia glomerata、红火蚁Solenopsis invicta和常见黄胡蜂Vespula vulgaris过敏原3的氨基酸序列一致性分别为50.44%、50%、48.89%和47.83%。荧光定量PCR结果表明,SgA3基因在雌成虫中高表达,且在毒液器官中的表达量显著高于雌成虫其他组织和雄成虫中的表达量。利用pSUMO-Mut表达载体,成功表达并纯化得到高纯度的SgA3重组蛋白。该研究结果为进一步研究SgA3基因的功能奠定了基础。

In order to investigate the physiological function of venom allergen 3 gene of Scleroderma guani(SgA3),this gene was cloned by RT-PCR.Its expression pattern at different developmental stages and in various tissues was analyzed by real time fluorescence quantitative PCR(qPCR).This gene was expressed by using prokaryotic vector pSUMO-Mut.The cloned ORF of SgA3 gene is 699 bp in length,encoding 233 amino acids.The amino acid positions 1~23 at the N-terminus of deduced amino acid of SgA3 gene was signal peptide sequence.Multiple sequence alignment analysis indicated that between SgA3 shared the amino acid sequence identity of 50.44%,50%,48.89%and 47.83%with allergen 3 of Nasonia vitripennis,Cotesia glomerata,Solenopsis invicta and Nasonia vitripennis,respectively.qPCR results showed that SgA3 gene was highly expressed at female adult stage and had higher expression in venom apparatus than in other female tissues and male adults.This venom gene was successfully expressed using vector pSUMO-Mut and high-purity SgA3 recombinant protein was obtained.The findings lay a foundation for further characterizing the physiological function of this venom gene.