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目的克隆获得腰果主要过敏原Ana o 3基因,并利用pCold-SUMO原核表达载体重组表达Ana o 3并鉴定免疫活性。方法提取腰果总RNA,逆转录至c DNA,设计特异性引物,通过巢式PCR技术克隆腰果Ana o 3基因,将其插入pCold-SUMO vector,鉴定并测序;将测序正确的阳性质粒转化大肠埃希菌BL21(DE3),低温15℃诱导表达。摸索诱导表达条件,经镍柱纯化,并通过Western blot鉴定免疫活性。结果测序结果表明,克隆腰果Ana o 3基因片段全长为417 bp,与GenBank上Ana o 3基因CDS序列基本一致;十二烷基硫酸钠聚丙烯酰胺凝胶电泳结果表明,目的蛋白分子量为27 kD左右,大小与理论值相符;Western blot结果表明,与腰果过敏阳性血清具有良好的反应性。结论从腰果中成功克隆了Ana o 3基因,并于原核系统表达了Ana o 3蛋白,证实了此蛋白与腰果过敏血清具有良好的反应性。
Objective To clone the Ana o 3 gene, a major caspian allergen, and use recombinant plasmid pCold-SUMO to express Ana o 3 and identify its immunological activity. Methods The total RNA of cashew nuts was extracted and reverse transcribed into c DNA. The specific primers were designed. Ana o 3 gene was cloned by nested PCR and inserted into pCold-SUMO vector for identification and sequencing. The correct positive plasmid was transformed into large intestine Corynebacterium BL21 (DE3), low temperature induced expression at 15 ℃. Induction expression conditions were explored, purified by nickel column, and immunological activity was identified by Western blot. Results The sequencing results showed that the full length of Ana o 3 gene was 417 bp, which was consistent with the CDS sequence of Ana o 3 gene in GenBank. The results of SDS - PAGE showed that the molecular weight of the target protein was 27 kD, the size of the theoretical value consistent; Western blot results show that, and cashew allergy positive serum has good reactivity. Conclusion The Ana o 3 gene was successfully cloned from cashew nuts and the Ana o 3 protein was expressed in the prokaryotic system. It was confirmed that this protein has good reactivity with cashew allergy serum.