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目的:构建人蛋白S(protein S,PS)的原核表达载体并诱导其表达。方法:自行设计引物,通过PCR技术,以人PS真核表达载体为模板扩增PS成熟肽编码序列,PCR产物经EcoR1/BamH1双酶切后,将其克隆至GST融合表达载体pGEX-2T中,在E.coli BL21中诱导GST-PS融合蛋白的表达。结果:聚丙烯酰胺凝胶电泳(10%SDS-PAEG) 显示,在IPTG的诱导下,BL21重组菌高效表达出一个分子量约为96kD产物,与预期大小相符,该产物可通过GST亲和层析柱纯化。对重组质粒的序列分析表明,插入片段的序列与Genebank登录的人PS基因编码序列完全一致。结论:人PS 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,为进一步研究奠定了基础。
Objective: To construct prokaryotic expression vector of human protein S (PS) and induce its expression. Methods: Primer was designed by PCR. The PS mature peptide coding sequence was amplified by PCR from human PS eukaryotic expression vector. After digested with EcoR1 / BamH1, the PCR product was cloned into GST fusion expression vector pGEX-2T , Induced the expression of the GST-PS fusion protein in E. coli BL21. Results: Polyacrylamide gel electrophoresis (10% SDS-PAEG) showed that the BL21 recombinant strain efficiently expressed a product with a molecular weight of about 96 kD under the induction of IPTG, which was consistent with the expected size. The product was purified by GST affinity chromatography Column purification. Sequence analysis of the recombinant plasmids showed that the sequence of the inserted fragment was identical to that of the human PS gene registered by Genebank. CONCLUSION: Human PS coding sequence has been cloned into GST fusion expression vector pGEX-2T and expressed in E. coli BL21, which laid the foundation for further study.