摘要
目的诱导表达可生物素化的人类白细胞抗原HLA-DRB1~*07:01蛋白,以制备MHC四聚体,为后续研究肽段与MHCⅡ的结合力、检测抗原特异性T细胞提供必要工具。方法从Gen Bank中获得HLA-DRB1~*07:01的c DNA序列,人工合成基因片段时,α链、β链的跨膜区加入亮氨酸拉链,其中β链还在跨膜区加入Avi标签。α链和β链分别与表达载体p ET-44a和p ETduet-1-Bira连接后转入大肠杆菌Rosetta构建原核表达体系,通过氨苄青霉素(Amp)抗性基因筛选,利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,获得目的蛋白,并用免疫印迹法分析鉴定目的蛋白。结果成功构建了重组质粒p ET-44a-HLA-DRB1~*07:01α链和p ETduet-1-HLA-DRB1~*07:01β链-Bir A,0.25 mmol/L的IPTG可高效诱导目的蛋白表达,α链可被HLA-DRA抗体特异识别,目的蛋白可生物素化且可被链霉素特异识别。目的蛋白主要以包涵体形式表达,经反复洗涤可获得高纯度的蛋白。结论成功构建了HLA-DRB1~*07:01蛋白的原核表达系统,并得到纯化的可生物素化重组表达蛋白,为进一步制备HLA-肽四聚体奠定了实验基础。
Objective To obtain a recombinant HLA-DRBl＊07 ： 01 protein for preparing HLA-DRB 1＂07 ： 01-peptide tetramers to detect antigen-specific CD4＋ T cells. Methods The eDNA sequence of HLA-DRBI＊07： 01 was obtained from GenBank. The gene fragment was synthesized. The leucine zipper was added to the transmem- brahe region of α chain and βchain, and the β chain was also added with the Avi tag in the transmembrane region. The α and β chains were ligated with the expression vectors pET-44a and pETduet-l-Bira, respectively, and then transformed into E. eoli Rosetta to construct a prokaryotic expression system. The ampieillin （Amp） resistance gene was screened and induced by isopropyl-β-D-thiogalactoside （IFFG） to obtain the target protein, and the target pro- tein was analyzed and identified by Western blotting. Results The recombinant plasmid pET-44a-HLA-DRB 1＊07 ： 01 α chain and pETduet-1-HLA-DRBI＊07：01 β ehain-BirA were successfully constructed. 0.25 mmol/L IPTG could efficiently induce the expression of the target protein. The ct chain can be specifically recognized by the HLA-DRA antibody, the target protein can be biotinylated, and can be specifically recognized by streptomycin. It is mainly expressed in inclusion body, and high purity target protein can be obtained after repeated washing. Conclusions The prokaryotie expression system of HLA-DRBl＊07 ： 01 protein was successfully constructed, and the purified biologically recombinant protein was obtained, which laid the experimental foundation for the further preparation of HLA-peptide tetramers.
作者
李敏英
李文
陶爱林
LI Minying;LI Wen;TAO Ailin(Guangdong Provincial Key Laboratory of Allergy ＆ Clinical Immunology/The State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China)
出处
《实用医学杂志》
CAS
北大核心
2018年第7期1080-1084,共5页
The Journal of Practical Medicine
基金
国家自然科学基金资助(编号：81373128)
国家重大科技专项重大课题任务(编号：2016ZX08011-005)
广州医科大学科研项目(编号：2015C19)
