重组人抑癌相关蛋白核苷二磷酸激酶A的原核表达

目的:为提高表达量,简化纯化工艺,获得可用于临床研究的重组人NDPK-A蛋白,构建带有6×His纯化标签的原核表达载体,优化表达条件获得产物高表达并鉴定产物活性。方法:将抑癌基因nm23-H1 从质粒pBVNMH1中亚克隆于带有纯化标签的表达载体pQE40中;梯度变化表达条件获得产物高表达;镍离子螯合层析柱纯化目的蛋白;Western blot鉴定产物的免疫原性;HPLC测定产物的激酶活性;鸡胚尿囊膜试验鉴定产物抑制血管新生的生物活性。结果:pQE-40中亚克隆的nm23-H1 序列无误;目的蛋白最高表达量可达49.6%;纯化的表达产物能与天然NDPK－A的多克隆抗体特异结合;酶比活为472U/mg;具有抑制鸡胚尿囊膜血管新生的活性。结论:表达质粒pQE-nm23H 1 能高效表达重组人NDPK-A,纯化工艺简便,产物活性与天然NDPK-A无异。

Expression of recombinant human tumor suppressor NDPK-A in E.coli

AIM: To construct E. coli expression plasmid of recombinant human NDPK-A with a 6×His tag, optimize the expression condition and identify the activity of the product. METHODS: nm23-H 1 was subcloned from plasmid pBVNMH1 to pQE40 which contain 6×His purification tag. The expression condition was modulated in grades to get the optimal expression. We purified protein with the Ni⁺-NTA affinity chromatography column, identified the immunogenicity of the product with Western blot, and measured the kinases activity with HPLC. In addition, angiogenesis inhibition activity of rhNDPK was identified by CAM. RESULTS: The sequence of nm23-H 1 subclone in pQE40 was exactly correct. The expression rate of rhNDPK-A was 49.6%. Purified rhNDPK-A specially recognized the antiserum of NDPK-A. It also inhibited angiogenesis. CONCLUSION: PQE-nm23H1 containing 6×His can express target protein at high level. This purification method is simple than other methods, and the product has the same activity as natural human NDPK-A.