【摘要】 目的:克隆乳胶主要过敏原Hevb3基因,构建其原核表达载体并表达和纯化重组蛋白,了解重组的Hevb3是否具有和相应IgE特异结合的过敏原特性。方法:用RT-PCR方法从新鲜海南橡胶树叶提取的总RNA中扩增出Hevb3的cDNA,将其克隆于原核表达载体pQE30质粒,转化大肠杆菌XL1-Blue,酶切和测序验证构建的载体,利用Ni-NTA柱层析纯化重组蛋白并用SDS-PAGE和WesternBlot验证。结果:酶切和测序结果证实克隆了正确的Hevb3序列,重组表达并纯化的蛋白具有和特异IgE抗体结合的过敏原特性。结论:成功构建了乳胶主要过敏原的原核表达载体,并纯化了具有过敏原特性的重组蛋白,为过敏原的标准化创造了条件。更多还原

【Abstract】 Objective:To construct a prokaryotic expressing vector and to purify recobinant from major allergen,Hev b3of latex,and to know if the recombinant protein has allergenicity.Meth-ods :Using reverse transcription polymerase chain reaction(RT-PCR),the Hev b3cDNA was cloned from the total RNA of rubber-tree leaf from Hainan,China.Then we incorporated the cDNA into pQE30plasmid.The expression of the recombinant Hev b3protein by the E.coli XL1-Blue was pu-rified using Ni-NTA column chromatography and analyzed by both SDS-PAGE and western blot test-ing.Results:The genetically engineered E.coli XL1-Blue did express the recombinant Hev b3pro-tein and had allergenicity,which was confirmed by the western blot.Conclusion:This successful con-struction of recombinant Hev b3protein will facilitate the allergen standardization by the way of re-combinant technique in China.更多还原