小鼠TSR2-3/TSP-1基因片段的克隆及其GST融合蛋白的表达和纯化

Cloning of mouse TSR2-3/TSP-1 gene segments and expression and purification of their coding protein fused with GST

目的:获取小鼠TSR2-3/TSP-1基因片段,并高效表达和纯化GST-TSR2-3融合蛋白。方法:应用RT-PCR技术,从小鼠肺总RNA中,获得编码TSR2-3的基因片功能片段,测序后,通过酶切克隆至表达载体pGEX-5X-2,构建重组表达载体,并导入E.coli BL21宿主菌中,IPTG诱导表达重组的GST融合蛋白,亲和层析纯化表达产物,SDS-PAGE和Western blot进行分析鉴定。结果:获得小鼠TSR2-3基因片段,测序结果与GenBank的基因序列一致,重组GST融合蛋白经SDS-PAGE和Western blot分析,在相对分子量39 kDa处,出现特异性蛋白条带,重组蛋白经GST亲和层析柱纯化后,得到了高纯度的融合蛋白。结论:成功克隆小鼠TSR2-3基因片段,并在E.coli BL21中高效表达,亲和层析后获得高纯度GST-TSR2-3融合蛋白。

Objective： To obtain mouse TSR2-3/TSP-1 segments, and to express the target protein efficiently in Escherichia coli （E. coli） BL21 and purify it. Methods： TSR2-3/TSP-1 gene segments were amplified by reverse transcriptase polymerase chain reaction （RT-PCR） from mouse lung total RNA, After sequenced, the reconstructed expression vectors were constructed by enzyme digestion and cloned into expression vector pGEX-5X-2. Then the reconstructed vectors were transformed into E.coli BL21. Recombinant GST fusion proteins were expressed by the induction of IPTG and purified through GST-affinity column. Results： The sequences of cloned mouse TSR2-3 gene segments were identical with those reported in GenBank. A protein band of 39 kDa appeared on SDS-PAGE gel after the expressed GST fusion protein was separated by SDS-PAGE. Tnen the high purity fusion protein was obtained. Conclusion： The mouse TSR2-3 gene segments could be successfully cloned and efficiently expressed in E. coli BL21, and the GST-fused target proteins with high purity could be obtained.