人Cosmc胞外段蛋白在昆虫细胞Sf-9中的表达与纯化研究

发布时间：2018-04-18 16:52

本文选题：人Cosmc胞外段 + 昆虫细胞　；参考：《中国免疫学杂志》2016年10期

【摘要】：目的:利用昆虫细胞Bac-to-Bac杆状病毒表达系统表达人Cosmc胞外段蛋白,为体外研究Cosmc蛋白的结构和功能奠定基础,同时也为O-连接糖基化及相关疾病的研究提供思路。方法:采用Bac-to-Bac系统,构建重组转移质粒p FastBac1-Cosmc extracellular domain(p Fast Bac1-Cosmc ED),转化到含穿梭载体Bacmid的感受态细胞DH10Bac中,通过蓝白斑筛选和PCR鉴定,获得重组转座子r Bacmid-Cosmc ED。在脂质体介导下,重组病毒感染Sf-9无血清细胞,在Sf-9中表达Cosmc胞外段蛋白,由于Cosmc N端加有昆虫细胞信号肽HBM(Honey Bee Melittin)且不含有跨膜段,所以Cosmc胞外段蛋白表达后分泌到培养基上清中,通过SDS-PAGE和Western blot对表达产物进行检测,并通过Ni-NTA亲和层析柱对其进行纯化分析。结果:SDS-PAGE和Western blot分析显示得到一条分子量约为33 k D的特异性条带,与目的蛋白大小相符,质谱结果进一步证明所得蛋白为Cosmc胞外段蛋白。结论:Sf-9细胞中可成功表达Cosmc胞外段蛋白,为进一步研究Cosmc的结构和功能奠定基础。
[Abstract]:Objective: to express human Cosmc extracellular segment protein by using insect cell Bac-to-Bac baculovirus expression system, and to lay a foundation for the study of the structure and function of Cosmc protein in vitro, and to provide a way for the study of O- linked glycosylation and related diseases.Methods: the recombinant transfer plasmid p FastBac1-Cosmc extracellular domain(p Fast Bac1-Cosmc was constructed by Bac-to-Bac system and transformed into the competent DH10Bac containing shuttle vector Bacmid. The recombinant transposon r Bacmid-Cosmc ED was obtained by blue and white spot screening and PCR identification.The recombinant virus was infected with Sf-9 serum-free cells mediated by liposome, and expressed extracellular segment protein of Cosmc in Sf-9. Due to the addition of HBM(Honey Bee Melittinin, an insect cell signal peptide was added to the N-terminal of Cosmc and did not contain transmembrane segments.So the extracellular segment protein of Cosmc was expressed and secreted into the supernatant of culture medium. The expression product was detected by SDS-PAGE and Western blot, and purified by Ni-NTA affinity chromatography.Results A specific band with molecular weight of about 33 KD was obtained by the analysis of Western blot and 7% SDS-PAGE, which was consistent with the size of the target protein. The result of mass spectrometry further confirmed that the protein was an extracellular segment of Cosmc.Conclusion the extracellular segment protein of Cosmc can be expressed successfully in Sf-9 cells, which lays a foundation for further study on the structure and function of Cosmc.
【作者单位】：上海交通大学医学院附属上海儿童医学中心风湿免疫科;
【基金】：上海市浦东新区科技发展基金创新资金资助(No.PKJ2012-Y51)
【分类号】：R593.2

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