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目的鉴定肝癌细胞株 SMMC-7721细胞膜上与β_2糖蛋白Ⅰ(β_2GP Ⅰ)特异结合的蛋白及其功能。方法应用自制的β_2GP Ⅰ亲和柱,将制备的肝脏细胞多聚核糖体提取液过柱,使β_2GP Ⅰ特异结合蛋白的多聚核糖体及其 mRNA 复合体结合其上,并洗脱收集。将洗脱液用 cDNA 合成试剂盒及 cDNA PCR 试剂盒处理得到相应的 DNA,并测序分析。逆转录 PCR 从肝细胞株 SMMC-7721中扩增膜联蛋白Ⅱ。流式细胞术检测膜联蛋白Ⅱ与β_2GP Ⅰ-GFP 对 SMMC-7721的竞争结合。结果获得的β_2GP Ⅰ特异结合蛋白 cDNA 片段约为1.1 kb,与人膜联蛋白Ⅱ具有高度同源性(98%)。SMMC-7721中有膜联蛋白Ⅱ的表达,并且1μl及4μl的膜联蛋白Ⅱ分别可使β_2GP Ⅰ-GFP 与 SMMC-7721的结合率由15.58%降至13.66%和7.56%。结论β_2GP Ⅰ在肝细胞膜上的受体是膜联蛋白Ⅱ,它可与β_2GP Ⅰ-GFP 竞争结合 SMMC-7721。β_2GP Ⅰ可能通过膜联蛋白Ⅱ介导 HBV 入侵肝细胞。
Objective To identify the specific binding proteins and their functions of β_2GP Ⅰ in the cell membrane of hepatocellular carcinoma cell line SMMC-7721. Methods The self - made β_2GP Ⅰ affinity column was used. The prepared polysaccharide extract of liver cells was passed through the column, and the polysaccharide of β_2GP Ⅰ specific binding protein and its mRNA complex were bound to it and eluted. The eluate was treated with cDNA synthesis kit and cDNA PCR kit to obtain the corresponding DNA, and sequenced and analyzed. Reverse transcription PCR was performed to amplify annexin II from hepatocyte cell line SMMC-7721. Flow cytometry was used to detect the competitive binding of annexin Ⅱ and β_2GP Ⅰ-GFP to SMMC-7721 cells. Results The cDNA fragment of β_2GP Ⅰ specific binding protein was about 1.1 kb, highly homologous to human annexin Ⅱ (98%). Annexin II was expressed in SMMC-7721, and the binding rate of β 2 GP Ⅰ-GFP to SMMC-7721 was reduced from 15.58% to 13.66% and 7.56% respectively by 1μl and 4μl annexin Ⅱ. Conclusion The receptor of β 2 GP Ⅰ on hepatocyte membrane is Annexin Ⅱ, which competes with β 2 GP Ⅰ-GFP for binding to SMMC-7721. β_2GP Ⅰ may mediate HBV invasion of hepatocytes by Annexin Ⅱ.