Objective To construct the SAP 2 prokaryotic expression vector, to express and purify solvable protein, so as to lay a foundation for antibody preparation and Sap2 antigen detection. Methods After the target gene fragment SAP 2 was obtained by standard PCR amplification, the SAP 2 and plasmids pMAL-c2x ( + ) were cleaved with two restriction endonucleases, and the digestion products were connected. Ligation products were transformed into competent cell, E. coli TOP10. Then positive clones of re-combined plasmid were screened and identified by DNA sequencing. After recombinant plasmid pMAL-c2x/SAP2 was transformed into E. coli strain BL21 ( DE3) which was induced by IPTG subsequently. The fusion protein was purified by Amylose Resin affinity chromatography, and then was cleaved by Xa factor. Results The target gene SAP 2 amplified by PCR had the same molecular size as predicted. It was inserted directionally into vector pMAL-c2x ( + ). Procaryotic expression plasmid pMAL-c2x/SAP2 can express soluble fusion protein MBP-Sap2 by IPTG induction 14 h later. Total 32 mg target protein Sap2 was obtained after purifying and cleaving label. Conclusion The recombined plasmid pMAL-c2x/SAP2 is successfully and highly expressed in BL21 (DE3) with soluble form. Target protein Sap2 was successfully obtained through affinity chromatography and proteinase cleavage.%目的 构建SAP2重组原核表达载体并表达、纯化出可溶性的蛋白,为抗体制备及Sap2抗原检测奠定基础.方法 提取白念珠菌基因组DNA为模板,经PCR方法获取SAP2目的基因.双酶切SAP2基因与原核表达载体pMAL-c2x(+),连接酶切产物,转化大肠杆菌TOP10感受态细菌,筛选菌落和测序鉴定.将pMAL-c2x/SAP2重组质粒转化大肠杆菌BL21(DE3)感受态细胞,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达出可溶性的融合蛋白,经直链淀粉树脂亲和层析、蛋白酶Factor Xa切割标签获得纯化的Sap2蛋白.结果 经PCR扩增获得正确的SAP2序列并定向插入原核表达载体pMAL-c2x(+)中.重组原核表达载体pMAL-c2x/SAP2经IPTG诱导14 h后表达出可溶性的融合蛋白,并经纯化、切除标签后得到目的蛋白.结论 成功构建了白念珠菌天冬氨酸蛋白酶原核表达质粒pMAL-c2x/SAP2,该质粒在BL21 (DE3)中可获得高效融合表达,通过亲和层析纯化及标签切割得到了氨基酸序列同天然蛋白一致的目的蛋白.
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