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目的:真核表达抗人乙酰胆碱受体单链抗体637(scFv637)与人血清白蛋白(HSA)的融合蛋白,以提高融合蛋白的产量和增强其生物学活性。方法:将携带有重组质粒的毕赤酵母菌GS115用G-418筛选高拷贝重组转化子克隆,诱导表达目的蛋白,表达产物用斑点杂交试验检测,并以SDS-PAGE鉴定融合蛋白的相对分子质量(Mr),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(AChR)的亲和性。结果:斑点杂交试验显示成功表达出目的蛋白,其Mr约为97400,在人肋间肌细胞膜之间有荧光出现。结论:在毕赤酵母菌GS115中成功表达出scFv637-HSA融合蛋白,并且融合蛋白可以与人肋间肌AChR结合,这为进一步研究融合蛋白的功能和应用奠定了基础。
OBJECTIVE: To eukaryoticly express the fusion protein of anti-human AChR-637 (scFv637) and human serum albumin (HSA) in order to increase the yield of the fusion protein and enhance its biological activity. Methods: The Pichia pastoris GS115 carrying the recombinant plasmid was screened by G-418 to clone the high-copy recombinant transformant. The expressed protein was detected by dot-blot hybridization and the relative molecular mass of the fusion protein was identified by SDS-PAGE (Mr), the indirect immunofluorescence technique was used to determine the affinity of the fusion protein with human intercostal muscle acetylcholine receptor (AChR). Results: Dot blot hybridization showed that the target protein was successfully expressed, Mr was about 97,400, and fluorescence appeared between human intercostal muscle cell membranes. CONCLUSIONS: The scFv637-HSA fusion protein was successfully expressed in Pichia pastoris GS115 and the fusion protein can bind to human intercostal AChR, which lays the foundation for further study on the function and application of the fusion protein.