摘要
目的构建致炎血管内皮细胞特异性靶向肽Nts-1的表达载体,表达、纯化融合蛋白,并对其靶向性进行初步验证。方法根据大肠埃希菌密码子偏好性,对Nts-1天然基因序列进行同义突变,并在Nts-1序列两端分别插入一个半胱氨酸使其环化后,克隆至p ET14b-EGFP载体中EGFP序列的C-末端,构建重组原核表达质粒p ET14b-EGFPNts-1,转化大肠埃希菌,IPTG诱导表达,表达的融合蛋白his-EGFP-Nts-1经纯化和SDS-PAGE鉴定后,用LPS致炎人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)体外验证其在细胞表面的亲和力。结果成功构建了p ET-14b-EGFP-Nts-1表达载体,纯化后的融合蛋白相对分子质量约32 000,纯度达95%以上。融合蛋白hisEGFP-Nts-1可特异性地靶向于致炎血管内皮细胞表面,其亲和力是由短肽Nts-1介导的。结论成功表达了融合蛋白his-EGFP-Nts-1,并初步验证了其对致炎血管内皮细胞的亲和力,为其生物化学活性及功能的研究奠定了基础。
Objective To construct the expression vector for peptide Nts-1 to express and purify the fusion protein and preliminarily verify its targeted binding to inflamed endothelial cells. Methods The original sequence of Nts-1 was processed with synonymous mutation according to the preference codon of E. coli, of which both C- and N-terminuses were added with a cysteine for cyclization. The sequence was inserted into the C-terminus of EGFP in vector pET-14b- EGFP. The constructed prokaryotic expression vector pET-14b-EGFP-Nts-1 was transformed to E. coli for expression under induction of IPTG. The expressed fusion protein his-EGFP-Nts-1 was purified and identified by SDS-PAGE, of which the affinity to LPS-inflamed human umbilical vein endothelial cells （HUVECs） was identified in vivo. Results Expression vector pET-14b-EGFP-Nts-1 was successfully constructed, and the expressed fusion protein, with a relative molecular mass of about 32 000, reached a purity of more than 95%. The fusion protein was targeted to the surface of inflamed HUVECs specifically, of which the affinity was mediated by Nts-1. Conclusion Fusion protein his-EGFP-Nts-1 was expressed successfully, of which the affinity to inflamed HUVECs was verified preliminarily. It laid a foundation of study on biochemical activity and function of the fusion protein.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第1期21-25,29,共6页
Chinese Journal of Biologicals
关键词
Nts-1
血管内皮细胞
靶向肽
表达载体
Nts-1
Endothelial cells
Peptide with targeted binding
Expression vector
