腰果主要过敏原Anao2基因克隆及原核表达

Cloning expression,purification and characterization of cashew nut allergen Anao2

摘要: 目的克隆腰果主要过敏原Anao2基因,并利用pMAL-c表达载体表达该蛋白。方法提取腰果总RNA,设计特异性引物,反转录-聚合酶链反应(RT-PCR)克隆腰果Anao2基因,将其反转录基因连入pMD18T sim-ple vector,提取质粒、双酶切、鉴定并测序;将测序正确的片段连入原核表达载体pMAL-c,将重组质粒转入BL21宿主表达菌中,丙基-β-D-硫代吡喃半乳糖苷诱导表达目的蛋白Anao2。结果测序结果表明克隆腰果Anao2基因片段全长为1 332 bp,编码443个氨基酸,与GenBank中蛋白序列完全相同;对获得的重组蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定,目的蛋白大小与理论值相符。结论成功克隆并表达了腰果过敏原Anao2。

Abstract: Objective To clone and express the gene of the major allergen Anao2 from cashewnut.Methods The open reading frame(ORF)of Anao2 was cloned and inserted into the expression vector pMAL-c.The vector was transformed into Escherichia coli BL21(DE3)and the protein expression was induced by isopro-pyl-,-D-thiogalcatopyranoside(IPTG).Results The cloned ORF containing 1 332 bp and encoding 443 amino acids was authenticated to be Anao2.The recombinant Anao2 protein induced by IPTG was consistent with the actual value.Conclusion Cashew recombinant Anao2 protein is obtained.