国家自然科学基金(39970291)和教育部青年骨干教师基金资助课题.
Projects supported by the National Natural Science Foundation of China (Grant 39970291), Young Teacher Financial Project of Education Ministry.
采用高效液相色谱法分离鉴定了红细胞膜蛋白质跨膜域的亲水性肽链,结果显示血型糖蛋白A(GPA)Lys101~Asp130与带3蛋白有相关性.为进行深入研究,采用RT-PCR方法从K562细胞中扩增了410 bp的GPA基因,分别克隆到酵母双杂交BD端表达质粒和杆状病毒转移载体上.同时,以含有带3蛋白全长基因的质粒为模板扩增了348 bp的带3蛋白C端基因,将其克隆到酵母双杂交AD端表达质粒.经酵母双杂交营养缺陷培养选择和β半乳糖苷酶检测证实GPA与带3蛋白之间存在相互作用.GPA表达产物分别经抗带3蛋白和抗GPA抗体进行蛋白质印迹分析,表明二者具有免疫交叉反应.上述结果表明带3蛋白与GPA在结构与功能上存在着密切联系.
The correlation of glycophorin A and band3 was confirmed by purification and identification of hydrophilic peptides of red blood cell transmembrane domain with high performance liquid chromatograghy(HPLC). Glycophorin A gene(410 bp) was amplified from K562 with RT-PCR and subcloned into the yeast BD hybrid vector pGBKT7 and baculovirus transfer vector pFASTBac. Glycophorin A and band3 C-terminus were co-transformed into the yeast strain AH109 and the interaction was identified by nutritional selection and β-glycotosidase activity detection. The GPA expression product in Sf9 cells was analyzed on Western blot with anti-GPA and anti-human band3 antibodies, and the result showed that they have immunologic cross-link reaction. All the results above confirmed the interaction between band3 and glycophorin A.
杜洪清,傅国辉,姜晓殊,龙晓宇,张振宇,杨宝峰,孔宪刚.带3蛋白C端和血型糖蛋白A相互作用及其抗原相关性的研究[J].生物化学与生物物理进展,2002,29(2):240-246
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