大肠癌相关基因ST13的原核表达及鉴定

Expression and Identification of Colorectal-cancer-related Gene ST13 in E.coli

目的 :原核表达大肠癌相关基因 ST1 3 ,并予以鉴定。方法 :将 ST1 3 c DNA经 PCR扩增和亚克隆 ,与 GST原核表达载体 p GEX-4T-2重组 ,转化 E.coli INVαF′菌表达 ,经 GlutathioneSepharose 4B亲和层析 GST-ST1 3融合蛋白 ,再以凝血酶切得到 ST1 3蛋白。结果 :限制性酶切和DNA测序表明 ,ST1 3 c DNA ORF正确克隆于 p GEX-4T-2多克隆位点。经 IPTG诱导表达的GST-ST1 3融合蛋白主要存在于可溶性上清 ,表达量占大肠杆菌总蛋白 2 0 %。经亲和层析 ,每升诱导菌回收融合蛋白 2 .5mg,纯度为 91 .3 %。 Western-blot证实 ,原核表达的 ST1 3蛋白、细胞中天然 ST1 3蛋白的 1 0 % SDS-PAGE表观分子量 ,约 50 k D。结论 :成功构建了 ST1 3基因原核表达质粒 ,并大量表达和纯化了 ST1 3蛋白。

Objective: To study the expression of Colorectal cancer related Gene ST13 in E.coli on its structure and function. Methods: ST13 cDNA amplified by PCR was ligated with expression vector pGEX 4T 2 and transformed E.coli INVαF′. The expressed GST ST13 fusion protein was purified by Glutathione Sepharose 4B affinity chromatography in a single step, and then ST13 protein was purified after thrombin digestion. Results: BamH 1 restriction analysis and DNA sequencing showed that ST13 cDNA ORF was inserted into the expression vector with correct sequence. The expressed GST ST13 fusion protein induced by IPTG was mainly in the soluble supernatant with a yield of 20 % of total bacterial proteins about 2.5 mg per liter of induced cultures, and a purity of 91.3 % after affinity chromatography. Western blotting indicated that the apparent molecular mass of the ST13 protein estimated by 10% SDS PAGE mobility was approximately 50 kD. Conclusion: We succeeded in constructing the expression plasmid of ST13 gene and acquiring ST13 protein in large amount and high purity.