重组NuBCP-9/Tumstatin(74-98)融合多肽的构建、表达和活性研究

Construction,expression and activity evaluation of recombinant NuBCP-9/Tumstatin (74-98) fusion polypeptide

目的:构建抗肿瘤融合肽NuBCP-9/Tumstatin(74-98)(简称NT)原核表达载体并转化大肠杆菌BL21(DE3),获得具有多靶点抗肿瘤活性的融合肽。方法:将抗肿瘤小肽NuBCP-9和Tumstatin(74-98)用柔性肽(G4S)3连接,根据大肠杆菌偏爱密码子,采用重叠延伸PCR技术扩增融合肽序列,并将其克隆至pET32a(+)质粒中,构建pET32a-NT表达载体,转化大肠杆菌BL21(DE3),诱导表达,SDS-PAGE分析目的蛋白表达量并优化表达条件,经亲和色谱、酶切和超滤等方法分离、纯化得到重组融合肽;采用MTT法测定融合肽对人脐静脉内皮细胞ECV304和人肺癌细胞A549的抑制活性影响。结果:成功构建了重组表达载体pET32a-NT,优化表达条件获得了25%的可溶性蛋白表达量。活性研究表明,在融合肽终浓度为20μmol/L时,对ECV304和A549的抑制率分别为60.8%和65.2%。结论:融合肽初步表现出抗肿瘤活性,为进一步药效药理学研究奠定了基础。

Aim:To construct a prokaryotic expression vector carrying NuBCP-9-tumstatin(74-98)(abbreviated as NT) gene and to obtain the fusion peptide with antitumor activity.Methods:Nucleotide sequences of antitumor peptides,NuBCP-9 and Tumstatin(74-98),were connected via a linker(G4S)3 based on biased codons of E.coli the fused NT gene was reconstructed using SOE PCR,and inserted into pET32a(+) vector,and transformed in E.coli BL21(DE3).After expression,the novel fusion peptide was purified through nickel-affinity c...