人血浆蛋白S成熟肽基因在大肠杆菌中的融合表达

目的 构建人血浆蛋白S的原核表达载体并诱导其表达。方法 自行设计引物，采用PCR法，以现有人血浆蛋白S真核表达载体为模板，扩增人血浆蛋白S成熟肽的编码序列。PCR产物经EcoRI和BamHI双酶切后，克隆至GST融合表达载体pGEX-2T中，在E．coli BL21中诱导GST-人血浆蛋白S融合蛋白的表达。结果 对重组质粒的序列分析表明，插入片段的序列与Gen Bank登录的人血浆蛋白S基因编码序列完全一致。10％SDS聚丙烯酰胺凝胶电泳显示，在IPTG的诱导下，BL21重组菌高效表达分子量约为96kD的产物．并可通过GST亲和层析柱纯化。结论 人血浆蛋白S编码序列已被克隆至GST融合表达载体pGEX-2T，并在E．coli BL21中获得表达。

GST fusion expression of the mature peptide of human protein S in E.coli

Objective To construct a prokaryotic vector for the expression of human protein S. Methods The coding sequence of protein S mature peptide was amplified from an existing recombinant plasmid (PS pcDNA3) with primers designed in our laboratory, and cloned into pGEX-2T after restrictive digestion with EcoRI and BamHI . GST-protein S fusion protein was expressed after induction by IPTG. Results Sequencing and restriction digestion of the recombinant plasmid revealed the existence of the coding sequence for protein S mature peptide. SDS-PAGE showed that a protein band of about 96 kD could be induced by IPTG in the recombinant bacteria harboring the plasmid. Conclusion The coding sequence of protein S mature peptide is introduced into the pGEX-2T plasmid and a GST-fused protein S mature peptide could be induced at a high level.