人Fcα/μR的表达与功能的初步研究

【作者】 杨丽君；

【导师】 张伟；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2008， 博士

【摘要】 人Fcα/μR是IgA和IgM的Fc受体,能与多聚体IgM/IgA结合,因此可能在系统免疫和粘膜免疫中都有重要的作用。其基因位于1号染色体1q32,毗邻其它FcR基因,包括Poly Immunoglobulin Receptor（pIgR）。经蛋白序列分析,Fcα/μR是Ⅰ型跨膜蛋白,其膜外区有三个结构域组成,一个短的EC1区、一个免疫球蛋白样结构域的EC2区和一个特殊的EC3区。EC2区与pIgR胞外第一个结构域（D1）同源性很高,预测是结合IgM/IgA的区域。据报道,人Fcα/μR表达在扁桃体生发中心的滤泡树突状细胞、肾远曲小管和小肠潘氏细胞。为了研究Fcα/μR的性质,需要得到正确折叠的受体,而原核细胞表达的Fcα/μR以包涵体形式存在,难以以复性方式得到正确折叠的可溶性受体。为此我们尝试了用酵母和哺乳细胞真核表达Fcα/μR。在毕赤酵母中分泌表达的人Fcα/μR的膜外区在C端带MycHis₆标签,可以用Ni柱亲和纯化。经Western Blot鉴定,得到多个特异性条带（32-47、50、80、140kDa）,我们推测32-47kDa条带是部分降解的目的蛋白,80kDa和140kDa条带是二聚体或多聚体的目的蛋白,这种预测还需进一步证实。用CHO细胞系瞬时表达Fcα/μR野生型、膜外区以及C端带EGFP标签的融合蛋白,经Western Blot鉴定表明,这三种蛋白在CHO细胞中都以单体形式表达。通过细胞免疫组化和激光共聚焦分析显示,Fcα/μR主要表达在CHO细胞的胞浆。另外,我们还尝试用G418筛选的方法建立稳定表达Fcα/μR的CHO细胞株,经细胞免疫组化分析,药压得到的细胞阳性率为40%左右。FACS检测显示,在不打孔和0.2%皂苷打孔的情况下,CHO表达的Fcα/μR都不能结合IgA/IgM。其原因可能是Fcα/μR在CHO细胞中仅以单体形式表达,无法以成熟的二聚体形式结合配体。人Fcα/μR与pIgR的基因定位相邻近,结合配体的结构域同源性高,且配体都是多体的IgA和IgM,因此Fcα/μR也可能在粘膜免疫中发挥作用。为了研究Fcα/μR在粘膜系统的表达情况及其与pIgR的相互关系,我们检测了人乳腺癌细胞系MCF-7Fcα/μR表达情况。用RT-PCR、FACS、细胞组化和免疫荧光的方法检测到MCF-7细胞上同时有Fcα/μR和pIgR的表达,且MCF-7能特异性结合多体IgA/IgM。用兔抗人Fcα/μR和兔抗人pIgR多抗在免疫荧光中可检测到MCF-7表达的Fcα/μR和pIgR。结论,本项工作用酵母和哺乳细胞两种真核系统表达了Fcα/μR。酵母的表达产物含Fcα/μR单体、多聚体及其部分降解产物,CHO细胞表达完整受体但是只以胞浆中表达的单体形式存在,这种形式的Fcα/μR不能与血清IgA/IgM结合。另外我们发现人乳腺癌细胞系MCF-7在RNA水平和蛋白水平都表达Fcα/μR和pIgR,且能特异性结合多体IgA和IgM,因此可以用来研究Fcα/μR和pIgR在粘膜免疫中的功能。更多还原

【Abstract】 Human Fcα/μR is the Fc receptor for Immunoglobulin A（IgA） and Immunoglobulin M （IgM）.The receptor is expressed on cell surface as a homodimeric protein and it can bind antibodies of both IgM and IgA isotypes and thus may play a pivotal role in systemic and mucosal immunity.Fcα/μR is a single gene-family member encoded on Chromosome 1q32 near several other FcR genes,including Poly Immunoglobulin Receptor（pIgR）.The predicted Fcα/μR is a typeⅠtransmembrane protein with a short EC1 domain,an Ig-like EC2 domain and a unique EC3 in the extracellular region.This EC2 contains a conserved motif with the first domain（D1） of pIgR,which was predicted as an IgA/IgM binding domain.It has been reported that human Fcα/μR is expressed on follicular dendritic cells （FDC） in tonsil germinal centers,proximal tubular epithelial cells in kidneys and Paneth cells in small intestinal crypts.In order to get right folded soluble Fcα/μR,we expressed the extracellular region of Fcα/μR using Pichia pastoris sysyem.A MycHis₆ tag was added at the C terminal and the fusion protein was purified by Ni affinity chromatography.We got several specific bands （32-47,50,80,140 kDa） identified by Western Blot.Because the theoretical size of the fusion protein is 50 kDa,we predicted the 32-47 kDa bands were degradation products and the 80,140 kDa bands were dimeric or polymeric proteins.We expressed three types of Fcα/μR in CHO cells:the wild type,the extracellular region and a fusion protein with EGFP at the C terminal.Western Blot analysis showed that Fcα/μR was expressed in CHO cells as monomers.IHC and confocal microscope analysis showed it was expressed in the cytoplasm of CHO cells.To determine the binding specificity of this receptor,we established a CHO transfectant stably expressing Fcα/μR, 40%of which were positive,analyzed by IHC.FACS showed that the Fcα/μR expressed by CHO cells could not bind IgA or IgM,no matter permeated by 0.2%saponin or not.It is reported that Fcα/μR was expressed on the Ba/F3 surface as a homodimeric protein and bound polymeric IgA/IgM.To get the functional Fcα/μR,we are about to establish a Ba/F3 transfectant stably expressing it.The adjacent gene location,high protein homology and same ligands of Fcα/μR and pIgR drive us to explore the relationships between them.We selected human breast cancer cell MCF-7.First we should figure out whether MCF7 express Fcα/μR.We used RT-PCR,flow cytometry,IHC analysis and immunofluorescence and found MCF-7 expressed both Fcα/μR and pIgR.Functional binding tests were done using FITC labeled human IgA/IgM and analyzed by flow cytometry.The tesults showed that MCF-7 bound poly IgA/IgM specifically.At the same time,we prepared polyclonal antibodies to the two receptors and they work in the immunofluorescence.In conclusion,we expressed the extracellular region of human Fcα/μR with a MycHis₆ tag at the C terminal using Pichia pastoris sysyem and purified the fusion protein by Ni affinity chromatography;expressed human Fcα/μR in CHO cells,and found it was in the cytoplasm of CHO cells as monomers;discovered the expression of Fcα/μR and pIgR on human breast cance MCF-7,and the specific binding of MCF-7 and IgA/IgM.更多还原