DeVito L D, Mason B, Schneck J, Margulies D H, Sollinger H W, Burlingham W J
Department of Surgery, University of Wisconsin, Madison.
Hum Immunol. 1991 Oct;32(2):125-33. doi: 10.1016/0198-8859(91)90109-m.
We engineered a fusion gene which encodes the alpha 1 and alpha 2 domains of HLA-A2 with the alpha 3 and truncated transmembrane domains of the murine class I-like protein Q10b, and transferred it into mouse L cells along with the gene for human beta 2-microglobulin (beta 2m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was noncovalently associated with the human beta 2m light chain. Native detergent-solubilized HLA-A2 and secreted rA2/Q10b proteins were found to be similar by: (a) the binding to mouse monoclonal anti-HLA antibodies in an ELISA; (b) the blocking of lysis of HLA-A2+ cells by human anti-HLA-A2,-B17, anti-HLA-A2,9,28, and anti-HLA-A2,28 cross-reactive group (CREG) antisera in a complement-dependent cytotoxicity assay; and (c) the ability when coupled to Sepharose to selectively purify HLA-A2,9,28 and HLA-A2,28 CREG-specific antibodies. Mouse L cells expressing rA2/Q10b produced as much as 2.5 micrograms protein per 10(6) cells/day, or 50- to 100-fold more antigen on a per cell basis than the level of HLA-A2 expressed by B-lymphoblastoid cell line or spleen cells. Thus rA2/Q10b represents a viable alternative to detergent-solubilized HLA-A2 for purification of anti-HLA-A2 antibodies and analysis of anti-HLA-A2 immune responses.
我们构建了一个融合基因，该基因编码HLA - A2的α1和α2结构域与小鼠I类样蛋白Q10b的α3和截短的跨膜结构域，并将其与人类β2 - 微球蛋白（β2m）基因一起转入小鼠L细胞。分泌的rA2/Q10b基因产物由一条分子量为42 kd的重链组成，该重链与人类β2m轻链非共价结合。通过以下方面发现天然去污剂溶解的HLA - A2和分泌的rA2/Q10b蛋白相似：（a）在ELISA中与小鼠单克隆抗HLA抗体结合；（b）在补体依赖性细胞毒性试验中，用人抗HLA - A2、- B17、抗HLA - A2,9,28和抗HLA - A2,28交叉反应组（CREG）抗血清阻断HLA - A2 +细胞的裂解；（c）与琼脂糖偶联时能够选择性纯化HLA - A2,9,28和HLA - A2,28 CREG特异性抗体。表达rA2/Q10b的小鼠L细胞每10^6个细胞每天产生多达2.5微克蛋白质，或在每个细胞基础上产生的抗原比B淋巴母细胞系或脾细胞表达的HLA - A2水平高50至100倍。因此，rA2/Q10b代表了一种可行的替代方案，可替代去污剂溶解的HLA - A2用于纯化抗HLA - A2抗体和分析抗HLA - A2免疫反应。
