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
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摘要: 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20 μmol/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。Abstract: 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 chromatography，Factor Xa digestion and ultrafiltration.Biological activity of the fusion peptide on ECV304 and A549 cells was evaluated by MTT assay. Results ：A prokaryotic expression system with NT gene was successfully constructed.The soluble fusion peptide was accounted for approximately 25% when induced by 0.5 mmol/L IPTG at 30 ℃ for 4 h.The purified fusion peptide could inhibit cell growth of ECV304 and A549 with inhibition rates of 60.8% and 65.2% at 20 μmol/L，respectively. Conclusion ：A novel fusion peptide with antitumor activity was cloned，expressed and purified.
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Keywords:
- NuBCP-9 /
- tumstatin /
- fusion polypeptide /
- antitumor /
- prokaryotic expression
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