摘要
目的构建及纯化生物素化的sH-2Kd-HBc复合物。方法以原核表达的sH-2Kd-BSP为重链,β2m为轻链,分别纯化后与H-2Kd限制性9肽(HBc131~139)在体外采用稀释法进行共折叠复性,然后在B irA酶作用下,对折叠产物进行生物素化,形成了生物素化的H-2Kd-HBc复合物。通过凝胶过滤层析法进一步纯化生物素化的复合物单体。结果原核表达质粒pET-H-2Kd-BSP的测序结果与GenBank所公布的序列一致。利用辣根过氧化物酶标记的亲和素对折叠复性及生物素化后的复合物进行检测,证明成功获得了生物素化的sH-2Kd-HBc复合物。结论获得了纯化的生物素化sH-2Kd-HBc复合物单体,为进一步在体外构建sH-2Kd-抗原肽四聚体及制备人工抗原提呈细胞,深入研究HBV感染过程中特异性CTL应答和效应机制奠定了基础。
Objective To prepare and purify biotinylated sH-2K^d-HBc complex. Methods Fusion proteins sH-2K^d-BSP and β2m were obtained by prokanyotical expression. After purification, the fusion proteins were refolded into sH-2K^d-HBc complex in the presence of an H-2K^d-restricted antigenic peptide by dilution method. The complex then was biotinylated by BirA enzyme and purified via the gel-filtration chromatography. Results The sequencing showed that the sequence obtained was 100% homologenic with U47329 in Gene Bank. The biotinylated sH-2Kd-HBc complex was confirmed by Western blotting with HRP-labled-avidin. Conclusion The obtained complex may offer a basis for further preparation of corresponding tetramer and artificial antigen presenting cells for the research of the mechanism of specific CTL responding and effecting.
出处
《医学分子生物学杂志》
CAS
CSCD
2006年第3期176-180,共5页
Journal of Medical Molecular Biology
基金
国家重点基础研究发展计划(973计划)(No.20014CB510008)~~
