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目的以膜联蛋白B1(AnxB1)为导向分子,与蜂毒素(MLT)基因融合,制备AnxB1-MLT融合蛋白,探讨其对磷脂酰丝氨酸脂质体的结合活性及对肝癌细胞SMMC7721和HepG2增殖的抑制作用。方法利用重叠延伸PCR技术构建AnxB1和MLT的融合基因AnxB1-MLT,克隆至原核表达载体pGEX-5T,转化至宿主菌K802,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导AnxB1-MLT融合蛋白表达,优化表达条件,用谷胱甘肽-S-转移酶(GST)亲和色谱纯化柱纯化融合蛋白。采用磷脂结合实验验证AnxB1-MLT融合蛋白的钙依赖性磷脂结合活性,采用CCK-8方法分别检测AnxB1-MLT融合蛋白对肝癌细胞系SMMC7721和HepG2细胞增殖的影响。结果 AnxB1-MLT融合蛋白在宿主菌K802中能够表达,在菌液生长至D600为0.6时,加入诱导剂IPTG至终浓度0.2mmol/L,低温(22~24℃)诱导表达4h,可使蛋白表达量较高且在上清有表达。通过GST亲和色谱纯化柱纯化融合蛋白,SDS-PAGE结果显示在63 000处可见单一目的条带,纯度>95%。AnxB1-MLT融合蛋白保留了AnxB1的钙依赖性磷脂结合活性,并能显著抑制肝癌细胞系SMMC7721和HepG2的增殖。结论成功制备了AnxB1-MLT融合蛋白,该融合蛋白具有AnxB1的钙依赖性磷脂结合活性,可抑制肝癌细胞SMMC7721和HepG2的增殖,为进一步利用动物实验研究AnxB1-MLT的抗肿瘤效果奠定了基础。
Objective AnxB1-MLT fusion protein was prepared by fusion with melittin (MLT) gene with annexin B1 (AnxB1) as a directing molecule, and its binding activity to phosphatidylserine liposomes and proliferation of hepatocellular carcinoma cells SMMC7721 and HepG2 Inhibition. Methods AnxB1-MLT fusion gene AnxB1-MLT was constructed by overlap extension PCR and cloned into prokaryotic expression vector pGEX-5T. The fusion protein was transformed into host strain K802 and induced by isopropyl-β-D-thiogalactoside The expression of AnxB1-MLT fusion protein was optimized and the fusion protein was purified by glutathione-S-transferase (GST) affinity chromatography. The phospholipid binding assay was used to verify the calcium-dependent phospholipid binding activity of AnxB1-MLT fusion protein. The effects of AnxB1-MLT fusion protein on the proliferation of hepatocellular carcinoma cell lines SMMC7721 and HepG2 were detected by CCK-8 assay. Results The AnxB1-MLT fusion protein was expressed in the host strain K802. When the growth of D600 was 0.6, the inducer IPTG was added to the final concentration of 0.2 mmol / L and induced by low temperature (22-24 ℃) for 4 h. The protein Higher expression level and expression in supernatant. The fusion protein was purified by GST affinity chromatography column. SDS-PAGE showed that there was a single band at 63 000 with a purity of> 95%. The AnxB1-MLT fusion protein retains the calcium-dependent phospholipid binding activity of AnxB1 and significantly inhibits the proliferation of hepatoma cell lines SMMC7721 and HepG2. Conclusion The AnxB1-MLT fusion protein was successfully prepared. The fusion protein has the calcium-dependent phospholipid binding activity of AnxB1 and inhibits the proliferation of hepatocellular carcinoma cells SMMC7721 and HepG2, which lays the foundation for further study on the anti-tumor effect of AnxB1-MLT in animal experiments.