Carachterization of B cell stimulatory factor capable of inducing autoantibody production and its receptor

1)用无蛋白RPMI1640培养的小鼠B151K12 T细胞杂交瘤细胞培养上清，用20-50%硫酸铵沉淀浓缩后，用DEAE-Sephadex A-50柱层析。利用B151-TRF2与GlcNAc残基(S)的特异结合能力，用GlcNAc共聚物(甲壳素)柱进一步纯化离子交换层析中的活性部分。洗脱液中B151-TRF2活性纯化约2000倍。与功能分析结果一致的是，利用纯化的标记了^&lt；125&gt；I的B151-TRF2组分的结合实验表明，B151-TRF2分子上没有检测到糖部分，B151-TRF2通过与B细胞表面末端的GlcNAc残基(S)相互作用而与B细胞结合。2)用抗末端GlcNAc残基的JL单抗(S)鉴定的B细胞亚群参与了B151-TRF2的应答。此外，还对可溶性m…进行了Western blotting分析用~(125)&gt；I-JL单抗获得较多的囊膜组分，可见四条相对分子质量约为25K、30K、30K、40K和60K的条带。3)从纯化的B151-TRF2超免疫大鼠脾细胞与P3U1骨髓瘤细胞融合建立的B细胞杂交瘤中，筛选出一株能产生特异性抑制B151-TRF2活性的单抗。包被纯化的5-6-9单抗的培养板(24W)能够吸附B151-TRF2的活性。4)B151-TRF2自身抗体产生的免疫遗传学研究表明，多克隆B细胞激活B151-TRF2诱导的小鼠红细胞(BrMRBC)抗体应答受H-2复合体I区基因控制。有趣的是，b细胞上表达的自身Ia识别特异性与体内BrMRBC特异性B细胞克隆的产生密切相关。这一发现对H-2连锁ir基因控制B细胞应答，特别是抗体谱的产生具有重要意义。较少

1) Culture fluid supernatant of murine B151K12 T-cell hybridoma grown in protein-free RPMI1640 medium was concentrated by 20-50% ammonium sulfate precipitation and applied onto DEAE-Sephadex A-50 column. The active fraction in the ionexchangechromatography was further purified with GlcNAc copolymer (chitin) column by taking advantage of the specific binding ability of B151-TRF2 to GlcNAc residue(s). B151-TRF2 activity in eluate fraction was purified approximately 2,000-fold. In consistent with the results of functional analysis, binding experiments utilizing purified B151-TRF2 fraction labelled with ^<125>I revealed that sugar moiety is not detected on B151-TRF2 molecule and that B151-TRF2 binds to B cells through interaction with their surface terminal GlcNAc residue(s). 2) B cell subpopulation identified by Jl monoclonal antibody (mAb) specific for terminal GlcNAc residue(s) was shown to be involved in the B151-TRF2 response. Furthermore, Western blotting analysis of SDS-solublized m … More embrane fraction with ^<125>I-Jl mAb revealed four bands with M.W of approximately 25K, 30K, 30K, 40K and 60K. 3) Among B cell hybridomas established by fusing P3Ul myeloma with spleen cells from rats hyperimmunized with purified B151-TRF2, we selected out a clone 5-6-9 which produces mAb capable of specifically inhibiting B151-TRF2 activity. Culture plate (24W) coated with purified 5-6-9 mAb was able to absorb B151-TRF2 activity. 4) Immunogenetic study on autoantibody production by B151-TRF2 demonstrated that in vitro anti-Bromelain treated mouse red blood cells (BrMRBC) antibody response of unprimed murine B cells induced by polyclonal B cells activation B151-TRF2 is controlled by I-region gene within H-2 complex. Interestingly, self-Ia recognitionspecificity expressed on b cells was closely associated with in vivo generation of BrMRBC-specific B cell clone. This finding has important implication to the problems of H-2-linked Ir-gene control of B cell response, especially generation of antibody repertoire. Less