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目的:制备负载SIV抗原肽的中国恒河猴Mamu-B*1703可溶性单体及其四聚体。方法:以含Mamu-B*1703重链cDNA序列的pMD19-T克隆为模板,通过PCR的方法克隆Mamu-B*1703重链基因,进而构建羧基端融合生物素化酶BirA底物肽(BSP)的Mamu-B*1703重链胞外域融合蛋白的表达载体,并在大肠杆菌中获得表达。β微球蛋白、SIV抗原肽共存时,通过稀释法复性可溶性Mamu-B*1703单体,经生物素化并纯化后与荧光素标记的链亲和素按4∶1的比例混合形成四聚体。结果:ELISA检测显示获得具有正确构象的负载SIV抗原肽的Mamu-B*1703四聚体。结论:印度恒河猴Mamu-B*1701特异性抗原肽IW9,与中国恒河猴的Mamu-B*1703相结合形成可溶性Mamu-B*1703/IW9单体和四聚体。
OBJECTIVE: To prepare Chinese rhesus Mamu-B * 1703 soluble monomers and their tetramers loaded with SIV antigen peptides. METHODS: Mamu-B * 1703 heavy chain gene was cloned by PCR using pMD19-T clone containing Mamu-B * 1703 heavy chain cDNA sequence as a template, and then a carboxyl terminal fused biotinylated BirA substrate peptide (BSP ) Mamu-B * 1703 heavy chain extracellular domain fusion protein expression vector and obtain the expression in E. coli. β microglobulin and SIV antigen peptide, the soluble Mamu-B * 1703 monomer was refolded by dilution method, biotinylated and purified, and then mixed with fluorescein-labeled streptavidin at a ratio of 4: 1 to form tetrakis Polymer. Results: The ELISA assay showed that Mamu-B * 1703 tetramer loaded with the SIV antigen peptide with the correct conformation was obtained. Conclusion: Mammalian Bovine Mammal-B * 1701 specific antigen peptide IW9, which binds to Mamu-B * 1703 in Chinese rhesus monkeys, forms soluble Mamu-B * 1703 / IW9 monomers and tetramers.