【摘要】 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。更多还原

【Abstract】 To express and identify fibronectin C-terminal heparin-binding domain(FNCH BD) polypeptides in Pichia pastoris expression system and study its function,the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector.After sequenced,the fragment was inserted into pAo815SM vector,and then cloned into the expression vector pPIC9k.The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115.The positive yeast clone was screened by G418 resistant,and the target protein was induced to express in the medium containing 0.5% methano1.The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography.The purified product was analyzed with mass spectrogram,SDS-PAGE,Western blotting and heparin affinity chromatography.The results showed that the target protein was around 32 kDa and the purity of the product was above 95%.FNCHBD could be specifically recognized by fibronectin polyclonal antibody.These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris,which provides a good strategy to further studies.更多还原