单链抗体637与人血清白蛋白融合蛋白的真核表达及功能鉴定

发布时间：2018-01-06 19:29

本文关键词：单链抗体637与人血清白蛋白融合蛋白的真核表达及功能鉴定　出处：《延边大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：将已经构建的重症肌无力抗乙酰胆碱受体主要免疫原区单链抗体637(ScFv)与人血清白蛋白(HSA)重组基因(ScFv-HSA)进行诱导表达，确定融合蛋白的分子量，检测融合蛋白与人肋间肌乙酰胆碱受体的亲和性。方法：将携带有重组基因表达载体的毕赤酵母菌GS115菌株复苏后，利用抗生素G-418筛选高拷贝重组子，用YPD培养基传代培养，再经BMGY、BMMY表达培养基培养3天，每24小时添加100％甲醇使甲醇终浓度为1％诱导表达。培养基上清液室温经14000g离心后，利用斑点杂交试验(dot blot)初步判定上清液中的融合蛋白；采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析融合蛋白的分子量；应用间接免疫荧光技术确定融合蛋白与人肋间肌乙酰胆碱受体的亲和性。结果：培养基上清液中有融合蛋白的表达，分子量约为97.4 KD，间接免疫荧光试验显示在人肋间肌细胞间有荧光出现。结论：已经从真核细胞表达载体成功表达出融合蛋白，并且此融合蛋白可以与人肋间肌乙酰胆碱受体结合。
[Abstract]:Objective: to construct the recombinant gene scFv-HSA-ScFv-HSAand the constructed single chain antibody (scFv) of the main immunogenetic region of myasthenia gravis against acetylcholine receptor (ACHR) and human serum albumin (HSA). The expression was induced. The molecular weight of the fusion protein was determined and the affinity of the fusion protein to the acetylcholine receptor of human intercostal muscle was determined. Methods: the recombinant gene expression vector of Pichia pastoris GS115 strain was resuscitated. High-copy recombinant clones were screened by antibiotic G-418 and cultured on YPD medium, and then cultured on BMGYYBMMY expression medium for 3 days. The final concentration of methanol was 1% when 100% methanol was added every 24 hours. The medium supernatant was centrifuged by 14000g at room temperature. The fusion protein in supernatant was preliminarily identified by dot blot assay. The molecular weight of the fusion protein was analyzed by SDS-PAGE with sodium 12 alkyl sulfate and polyacrylamide gel electrophoresis. Indirect immunofluorescence technique was used to determine the affinity of the fusion protein to the acetylcholine receptor in human intercostal muscle. Results: the fusion protein was expressed in the supernatant of culture medium with a molecular weight of 97.4 KD. Indirect immunofluorescence assay showed the presence of fluorescence in human intercostal muscle cells. Conclusion: fusion protein has been successfully expressed from eukaryotic expression vector. And the fusion protein can bind to acetylcholine receptor in human intercostal muscle.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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