人内皮抑素-白蛋白融合蛋白在毕赤酵母中的表达与鉴定  

Construction and expression of the endostatin-HSA fusion protein in Pichia pastoris

作　　者：赵媛媛[1,2] 吕茂民[1] 杨梅[1] 冯晶晶[1] 章金刚[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850 [2]解放军后勤指挥学院

出　　处：《中国输血杂志》2010年第4期255-258,共4页Chinese Journal of Blood Transfusion

摘　　要：目的构建编码人内皮抑素-白蛋白的融合基因,并在毕赤酵母(pichia pastoris)中获得高效表达。方法通过重叠延伸PCR的方法 ,构建编码人内皮抑素-白蛋白的融合基因,并克隆至pGEM-T载体中,测序正确后,将其亚克隆至酵母表达载体pPIC9中,然后将鉴定正确的重组表达载体pPIC9/ES-HSA线性化后转化入GS115宿主菌。采用原位双膜法筛选高表达菌株,对高表达菌株进行扩大培养,将表达上清液盐析后分别经阳离子交换层析、阴离子交换层析和亲和层析纯化,获得了重组融合蛋白,采用MTT法测定融合蛋白中内皮抑素的活性。结果成功构建了人内皮抑素-白蛋白融合基因和重组表达质粒pPIC9/ES-HSA,并筛选到表达较为理想的重组菌株pPIC9/ES-HSA/GS115,经甲醇诱导表达后纯化,融合蛋白的纯度达到92%以上,并具有抑制人血管内皮细胞系ECV-304细胞的增殖活性。结论本方法可成功获得具有人内皮抑素生物活性的重组人内皮抑素-人血清白蛋白融合蛋白。Objective To construct the recombinant plasmid which encodes human endostatin and human serum albumin fusion protein, and to obtain high express level of it in Pichia pastoris. Methods The two sequences of ES and HSA were ligated by overlap PCR extension, and then the fusion gene was cloned into the vector of pGEM-T. After ensuring the sequencing was correct, we cloned it into the expression vector pPIC9 to obtain the recombinant plasmid pPIC9/ES-HSA, and then the linearized plasmid was electrotransformated into GS115. The expressing strain was screened by the immunological based double filters screening method and then enlarged its cultivation. The aim protein of culture supernatant after salting-out, cation exchange chromatography, anion exchange chromatography and affinity chromatography, the activity of endostatin in fusion protein was detected via MTT.Results The recombinant expressing plasmid pPIC9/ES-HSA and recombinant strain of high-level expressing pPIC9/ES-HSA/GS115 were obtained. The expressing supernatant was purified by methanol induced, and the purity of fusion protein reached 92%. It can inhibit the proliferation activity of ECV-304. Conclusion The fusion protein of human endostatin and human serum albumin which have bioactivity of endostatin was obtained successfully.

关 键 词：人内皮抑素 人血清白蛋白 毕赤酵母 

分 类 号：R457[医药卫生—治疗学] Q813.2[医药卫生—临床医学]