Gabriel S E, Racette K J, Gaspar K J, Forsyth G W
Veterinary Physiological Sciences, University of Saskatchewan, Saskatoon, Canada.
J Membr Biol. 1992 Sep;129(3):323-8. doi: 10.1007/BF00232913.
Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (> 110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass > 100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.
用在小鼠体内产生的抗体来尝试鉴定负责传导性氯离子转运的蛋白质，这种转运可在猪回肠刷状缘膜囊泡中检测到。通过制备性SDS聚丙烯酰胺圆盘凝胶电泳，将猪的回肠刷状缘膜囊泡蛋白分离成五个不同分子量的组分。分离出的蛋白质组分用于免疫小鼠。通过蛋白质免疫印迹法筛选抗体与抗原的反应性，以及其对回肠刷状缘膜囊泡中传导性氯离子转运的影响。用组分I的刷状缘蛋白（> 110 kDa）免疫产生的多克隆抗血清能特异性抑制回肠刷状缘囊泡制剂对氯离子摄取的传导成分。抗原的蛋白质免疫印迹显示存在几种分子量> 100 kDa的蛋白质，它们可被免疫血清识别。将产生抑制传导性氯离子转运抗血清的小鼠的脾细胞与骨髓瘤细胞系融合。所得杂交瘤集落产生的抗体通过蛋白质免疫印迹分析与至少七条不同的蛋白带发生反应，并抑制刷状缘膜囊泡中的氯离子传导。
