摘要
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)Lys10 1～Asp130与带 3蛋白有相关性 .为进行深入研究 ,采用RT PCR方法从K5 6 2细胞中扩增了 410bp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 34 8bp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 pGBKT(7) 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 beta-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.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2002年第2期240-246,共7页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金 ( 39970 2 91)
教育部青年骨干教师基金资助课题~~
