腰果主要过敏原Ana o 3的重组表达与鉴定  

Prokaryotic expression and identification of recombinant cashew nut allergen Ana o 3

作　　者：闫娟娟[1] 佘钿田 张嘉懿[2] 边颖[1] 李会强[1] 

机构地区：[1]天津医科大学医学检验学院,天津300203 [2]天津市儿童医院检验科,天津300074

出　　处：《中国食品卫生杂志》2017年第1期37-41,共5页Chinese Journal of Food Hygiene

基　　金：天津医科大学校基金面上项目(2014KYM08)

摘　　要：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 and express an important cashew nut allergen Ana o 3 in Escherichia coli（ E. coli）using pCold-SUMO expression vector and identify its immunocompetence. Methods Total RNA was extracted from the cashew nut and reverse transcribed into c DNA. Then,the full-length cDNA sequence of Ana o 3 was amplified by nested polymerase chain reaction（ PCR） using specific primers,and subsequently inserted into the pCold-SUMO expression vector between StuⅠand Bam HⅠ restriction sites. The correct construct was identified by both colony PCR and gene sequencing.The inducible expression of Ana o 3 was performed at 15 ℃ by an addition of L-（ ＋）-arabinose and isopropyl b-dthiogalactoside（ IPTG）,and the purification of the recombinant His-tagged protein was accomplished by the Ni-NTA purification system. The immunocompetence of the recombinant Ana o 3 was evaluated by Western blot. Results DNA sequencing analysis showed that the full length of Ana o 3 was 417 bp,encoding a polypeptide of 138 amino acids which was in accordance with that in Gen Bank. Also,the sodium dodecyl sulfate polyacrylamide gel electrophoresis result indicated the molecular weight of the recombinant Ana o 3 was 27 kD,which was in agreement with the theoretical value.Additionally,Western blot results showed that the recombinant Ana o 3 had good reactivity with 13 cases of cashew-allergic serum samples. Conclusion A recombinant expression vector of Ana o 3 had been successfully constructed,and the purified recombinant protein exhibited good reactivity with cashew-allergic sera.

关 键 词：腰果 ANA O 3 基因克隆 重组 表达 过敏原 免疫印迹 食品安全 

分 类 号：R155[医药卫生—营养与食品卫生学]