In-fusion技术构建表达、纯化钠泵α3亚单位膜外区截断性片段重组质粒  

Expression and Purification of Truncated Fragment of Extracellular Segment of Sodium Pump α3 Subunit in Escherichia coli by In-fusion Technology

作　　者：张明娟[1] 杨军[2] 朱参战[1] 段宗明[1] 牛小麟[1] 王蓉[1] 宋雅燔[1] 

机构地区：[1]西安交通大学医学院第二附属医院心内科,西安710004 [2]西安交通大学医学院第二附属医院病理科

出　　处：《四川大学学报（医学版）》2009年第2期203-207,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30500204)资助

摘　　要：目的构建重组pGEX-6P-1原核表达载体，表达和纯化大鼠钠泵α3亚单位M1～M2和M3～M4膜外区的截断性片段。方法利用In-fusion技术将钠泵α3亚单位M1～M2和M3～M4膜外区基因片段插入线性化的pGEX-6P-1载体中，转化，提取质粒，PCR鉴定；用阳性重组质粒pGEX-Trf-α3转化大肠杆菌，IPTG诱导、表达融合蛋白GST—Trf-α3。采用Glutathione Sepharose 4B亲和层析纯化融合蛋白，SDS-PAGE电泳分析表达产物的可溶性及含量。采用放射性配体结合法检测其生物活性。结果PCR和基因测序分析显示大鼠钠泵a3亚单位M1～M2和M3～M4膜外区被成功插入pGEX-6P-1载体GST基因3’端，蛋白序列分析表明GsT-Trf-α3融合蛋白总长度262个氨基酸，理论相对分子质量应约为33．22×10^3。IPTG诱导后，GST-Trf-α3融合蛋白的表达量为10％，多以可溶性形式存在，可溶性蛋白表达量为80．8％。SDS-PAGE结果显示其纯度〉95％。生物活性实验结果显示GST—Trf-α3融合蛋白与^3H-哇巴因具有一定的结合能力，但结合能力较弱。结论采用In-fusion技术成功构建了表达GST-Trf-α3融合蛋白的原核表达载体，建立了纯化方法，获得高纯度的融合蛋白，为钠泵α3亚单位M1～M2和M3～M4膜外区功能以及其潜在的药理学作用研究获得了实验数据。Objective To construct prokaryotic expression system for expressing, purifying and identifying truncated fragment of extra-cellular segment of sodium pump α3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology. Methods According to the conservative sequence of M1-M2 and M3-M4 extra-cellular gene fragments of sodium pump α3 subunit, which published in GenBank, a serial of primers and gene fragments was designed, and directly synthesized to fuse the above two gene fragments. The fusion gene was fused with gene-specific primers by PCR, and then fusion gene fragment was fused into the single stranded homology regions of vector pGEX-6P-1 by in-fusion cloning to construct recombinant vector pGEX-Trf-α3 （Truncated fragment of extracellular segment of sodium pump α3 subunit, Trf-α3）. After DH10bac was transferred with it, the pGEX-Trf-α3 plasmid was purified and identified by PCR and sequenced. Then the recombinant plasmid pGEX- Trf-α3 was expressed in E. coli BL21 cells, inducted by IPTG.GST-Trf-α3 fusion protein was purified with Glutathione Sepharose 4B purifying system and analyzed by SDS-PAGE. Results The results of PCR and sequencing demonstrated that the M1-M2 and M3-M4 extra-cellular gene was inserted in plasmid pGEX-6P-1 vector successfully. And the sequence was correct. Protein sequence analysis showed that the GST-Trf-α3 fusion protein was consisted of 262 amino-acid residues. Relative molecular mass in theory was 33. 22 × 10^3. The amount of recombinant protein was 10% of the total bacteria protein. The soluble fusion protein was about 80. 8%. After affinity purification, the purity of GST-Trf-α3 fusion protein was over 95%. There was some extent binding activity between GST-Trf-α3 fusion protein and ouabain, but the activity was very low. Conclusion Prokaryotic expression system for expressing truncated fragment of extra-cellular segment of sodium pump a3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology had been co

关 键 词：钠泵α3亚单位 截断性片段GST融合基因表达系统 

分 类 号：Q78[生物学—分子生物学]