Lu J, Willis A C, Sim R B
Department of Biochemistry, University of Oxford, UK.
Clin Exp Immunol. 1993 Dec;94(3):429-34. doi: 10.1111/j.1365-2249.1993.tb08213.x.
In this study, we used human tonsils for the isolation of the 60 kD component of the Ro/SSA autoantigen, following the method described by Wu et al. (J Immunol Methods 1989; 121:219-24). Western blot analyses were carried out using Ro/SSA-reactive human Sjögren's syndrome sera, to follow the autoantigen through the purification procedure. A 60 kD Ro/SSA component was eluted as a broad peak from a Mono Q column. Within this peak, a much more abundant protein, co-migrating with the Ro/SSA component on SDS-PAGE, was also eluted. The more abundant protein was further purified on a Superose 12 column and its N-terminal sequence was shown to be identical to that of human calreticulin. The 60 kD Ro/SSA autoantigen was also further purified on the Superose 12 column and was eluted as an asymmetric peak, with the majority being eluted at a position corresponding to 60 kD, whereas the calreticulin-like protein was eluted from the same column as an apparent dimer of approximately 120 kD. A panel of five Ro/SSA-reactive human sera reacted with the purified Ro/SSA antigen, but not with the calreticulin-like protein. Therefore, it is clear that the calreticulin-like protein is not a Ro autoantigen and is distinct from the 60 kD Ro/SSA antigen. As the calreticulin-like protein is a much more abundant protein than the 60 kD Ro/SSA component, its co-purification with the autoantigen on ion-exchange and its close migration with the autoantigen on SDS-PAGE may explain why peptide sequences for human calreticulin were derived from apparent 60 kD Ro/SSA antigen preparations.
在本研究中，我们按照Wu等人（《免疫学方法杂志》1989年；121:219 - 24）所述的方法，使用人扁桃体来分离Ro/SSA自身抗原的60 kD组分。使用与Ro/SSA反应的人类干燥综合征血清进行蛋白质印迹分析，以追踪自身抗原在纯化过程中的情况。一个60 kD的Ro/SSA组分从Mono Q柱上以宽峰形式洗脱下来。在这个峰内，还洗脱了一种在SDS - PAGE上与Ro/SSA组分共迁移的更为丰富的蛋白质。这种更为丰富的蛋白质在Superose 12柱上进一步纯化，其N端序列显示与人钙网蛋白相同。60 kD的Ro/SSA自身抗原也在Superose 12柱上进一步纯化，并以不对称峰形式洗脱，大部分在对应60 kD的位置洗脱，而钙网蛋白样蛋白从同一柱上以约120 kD的明显二聚体形式洗脱。一组五种与Ro/SSA反应的人类血清与纯化的Ro/SSA抗原反应，但不与钙网蛋白样蛋白反应。因此，很明显钙网蛋白样蛋白不是Ro自身抗原，且与60 kD的Ro/SSA抗原不同。由于钙网蛋白样蛋白比60 kD的Ro/SSA组分丰富得多，它在离子交换时与自身抗原共纯化以及在SDS - PAGE上与自身抗原紧密迁移，这可能解释了为什么人钙网蛋白的肽序列来自明显的60 kD Ro/SSA抗原制剂。
