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为了深入研究血小板胶原受体糖蛋白Ⅵ(glycoprotein Ⅵ,GPⅥ)功能及筛选特异性抑制剂,利用基因重 组技术体外表达GPⅥ胞外区片段。采用PCR方法扩增GPⅥ胞外区片段cDNA,构建表达载体pET-20b(+)-GP Ⅵ,转化大肠杆茵BL21(DE3)pLysS,经IPTG诱导表达。表达产物经Ni-NTA Resin纯化、复性;使用Western blot方 法鉴定重组蛋白性质;采用胶原结合试验测定重组蛋白的胶原结合能力。结果表明,经测序证明PCR扩增产物与 GPⅥ胞外区cDNA序列完全一致;酶切分析证明成功构建了表达载体pET-20b(+)-GPⅥ;原核细胞经诱导表达后 出现新的相对分子量32 kD蛋白条带,诱导产物以包涵体形式存在;Western blot分析表明重组蛋白可与抗Penta- His抗体和抗GPⅥ多抗特异性结合;胶原结合试验表明重组蛋白拥有较好的生物学功能。结论:正确构建了GPⅥ 表达载体并成功表达重组蛋白,纯化、复性后的重组蛋白具有良好的抗原性和生物学活性。
In order to further study the function of platelet collagen receptor glycoprotein Ⅵ (GPVI) and to screen specific inhibitors, the extracellular region of GPVI was expressed in vitro using gene recombination technique. The cDNA of extracellular domain of GPVI was amplified by PCR. The expression vector pET-20b (+) - GP Ⅵ was constructed and transformed into E. coli BL21 (DE3) pLysS and induced by IPTG. The expressed product was purified by Ni-NTA Resin and refolded. The nature of the recombinant protein was identified by Western blot. The collagen binding ability of the recombinant protein was measured by collagen binding assay. The results showed that the PCR products were completely identical to the extracellular domain of GPVI by sequencing. The digestion analysis showed that the recombinant plasmid pET-20b (+) - GPⅥ was successfully constructed. After prokaryotic expression, a new relative molecular weight of 32 kD protein bands. The induced products existed as inclusion bodies. Western blot analysis showed that the recombinant protein could specifically bind to anti-Penta-His antibody and anti-GPVI polyketide. Collagen binding assay showed that the recombinant protein has good biological function. CONCLUSION: GPVI expression vector is constructed correctly and recombinant protein is expressed successfully. The purified and renatured recombinant protein has good antigenicity and biological activity.