肝细胞膜上β_2糖蛋白Ⅰ受体的鉴定

Identification of the β_2GP I-binding receptor on hepatocyte membrane

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 identified theβ2-glycoproteinI （β2GPI）-bound receptor on the membrane of hepatocellular carcinoma cells, and to analyze its function. Methods Through the β2GPI-affinity chromatography column, the peptide-polysome-mRNA complex specially binding to β2 GPI stayed with the column and was separated from the whole polysome of liver cells. Then it was eluted and collected. With the cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexin Ⅱ , and flow cytometry was used to study the competitive binding of annexin Ⅱ with β2GPI to SMMC-7721 cells. Results 1.1 kb cDNA fragment of the specific binding protein of β2GPI on liver cell membrane was obtained. And the sequence of cDNA shared a high homology with human annexin Ⅱ （98%）. Annexin Ⅱ was expressed on the membrane of the hepatocellular carcinoma cells of the line SMMC- 7721, and 1 μl and 4 μlof annexin Ⅱ caused the binding rate of β2GP I -GFP with SMMC-7721 cells from 15.58% to β.66% and 7.56% respectively. Conclusion The receptor of β2GPI on the membrane of hepatocellular carcinoma cells is annexin Ⅱ, and β2GPI may help HBV invade hepatic cells through combing with annexin Ⅱ on the liver cell membrane.