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为在毕赤酵母中表达纤维连接蛋白C端肝素结合域(Fibronectin C-terminal heparin-binding domainFNCHBD)多肽并研究其功能,通过PCR技术扩增FNCHBD目的基因,将目的基因与T载体连接,经测序正确后,插入pAo815SM酵母表达载体增加基因拷贝数,然后酶切克隆入酵母表达载pPIC9K;将重组质粒Sal I酶切线性化后转化毕赤酵母菌株,筛选工程菌,经甲醇诱导表达,用SDS-PAGE检测发酵上清液,表明有重组蛋白FNCHBD多肽的高表达,表达产物通过离心、超滤、离子交换层析纯化,纯化产物通过SDS-PAGE、Western blotting印迹、质谱及肝素亲和层沉析对表达产物进行鉴定。结果表明利用酵母工程菌成功表达和纯化了FNCHBD多肽,多肽的分子量接近32 kDa,纯化产物的纯度可达95%以上,能被FN多克隆抗体特异识别且具有多肽肝素结合活性,为后续结构及功能的研究奠定基础。
To express Fibronectin C-terminal heparin-binding domain (FNCHBD) polypeptide in Pichia pastoris and study its function, the target gene of FNCHBD was amplified by PCR and ligated with the T vector. After sequencing After correct insertion of pAo815SM yeast expression vector to increase the gene copy number, and then digested cloned into the yeast expression vector pPIC9K; recombinant plasmid Sal I digested linearized Pichia pastoris strains screened engineered bacteria, induced by methanol, SDS -PAGE detection of fermentation supernatant, indicating that the recombinant protein FNCHBD polypeptide is highly expressed, the expression product was purified by centrifugation, ultrafiltration, ion exchange chromatography, purified product by SDS-PAGE, Western blotting blotting, mass spectrometry and heparin affinity chromatography Analysis of the expression product identification. The results showed that the FNCHBD polypeptide was successfully expressed and purified by yeast engineering bacteria. The molecular weight of the polypeptide was nearly 32 kDa. The purity of the purified product was more than 95%. It could be specifically recognized by FN polyclonal antibody and had the activity of polypeptide heparin binding. Functional research laid the foundation.