蛔虫过敏原ABA-1融合基因在大肠杆菌中的表达及鉴定

Expression and identification of chimeric protein truncated from Ascaris allergen ABA-1

背景:利用蛔虫基因融合技术,可将过敏原ABA-1的主要基因进行融合。目的:利用大肠杆菌表达系统重组表达蛔虫主要过敏原ABA-1融合蛋白。方法:从Gene Bank和Protein Database中获取蛔虫主要过敏原ABA-1基因和蛋白序列,选定其中的ABA-1B1,ABA-1A2与ABA-1A1基因重组为融合基因BAA,经密码子优化后,全基因合成目的基因BAA并构建于表达载体PET-44a上,经KCM法转化入大肠杆菌JM109中进行克隆,PET-44a/BAA经NdeⅠ和PstⅠ双酶切及DNA测序正确后,转化入大肠杆菌Rosetta Blue TM中,经IPTG诱导表达。表达蛋白经Ni柱亲和层析纯化后,通过Western blot和氨基酸测序鉴定目的蛋白。结果与结论:大肠杆菌的融合蛋白BAA表达量约占总蛋白的40%,纯化后蛋其白纯度可达90%左右。Western Blot结果显示在相对分子质量45000处可见一特异目的条带,氨基酸测序显示N端15个氨基酸与目的蛋白完全相同。结果证实,融合蛋白BAA在大肠杆菌中得到高效的表达。

BACKGROUND：Serious allergic reaction could be caused by Ascaris anaphylactogen ABA-1 protein （Ascaris Body fluid allergen 1）.OBJECTIVE：To express the chimeric protein truncated from Ascais major allergen ABA-1 in Escherichia coli.METHODS：The chimeric gene,obtained from GeneBank and Protein Database and named as BAA hereafter,was constructed by tailoring the coding fragments of Ascaris main allergen ABA-1,i.e.,ABA-1B1,ABA-1A2 and ABA-1A1.The fusion gene BAA was synthesized and cloned into expression vector PET-44a,and transformed into Escherichia coli JM109 with the method of KCM.When the plasmid was confirmed by double restriction enzymatic digestive test with NdeⅠand PstⅠand DNA sequencing,it was transformed into Escherichia coli RosettaBlue^TM step by step.The produced chimeric protein BAA was purified by nickel ion affinity chromatography,followed by identification by Western Blot and protein sequencing.RESULTS AND CONCLUSION：Chimeric protein was inducibly expressed in Escherichia coli,with the production up to 40% of the total protein.Protein purity could reach about 90% after purification.Western Blot showed a specific band at 45 000,and amino acid sequencing identified that the 15 N-terminal amino acids were identical to the target protein.The chimeric protein has been efficiently expressed in Escherichia coli.