蜂毒肽基因原核表达载体的构建及重组蛋白生物活性作用的研究

Study on construction of prokaryotic expression vector of melittin gene and bioactivity of recombinant protein

本研究根据Genebank中公布的蜂毒肽(melittin)基因序列(NM001011607),将其插入pGEX6p-1载体,转入BL21(DE3)进行诱导表达并纯化重组蛋白melittin,测定其浓度,采用SDS-PAGE电泳及免疫印迹分析重组蜂毒肽的分子量和活性;进行抑菌试验,以及肿瘤细胞毒性实验,判断重组蜂毒肽的生物活性。重组蛋白经SDS-PAGE和灰度分析在25~35 kDa之间出现一条明显条带;重组蛋白对金黄色葡萄球菌有高度敏性,对大肠埃希菌有中度敏感性,对铜绿假单胞菌和伤寒沙门菌有低度敏感性。5个浓度重组蛋白50、25、12.5、6.25、3.125 mg/L分别作用人乳腺癌MCF-7细胞,在12、24、36、48、72 h时间对人乳腺癌MCF-7细胞生长均有稳定抑制作用,与对照组相比有明显差异(P<0.05)。蜂毒肽融合蛋白通过pGEX6p-1载体转化到原核细胞进行表达稳定可行,且具有明显抑菌和抗肿瘤的作用。

Gene fragments of melittin were synthesized according to the sequence of the bee venom gene(melittin)published in Genebank(NM001011607).It was inserted into pGEX6p-1 vector and transferred into BL21(DE3)to induce expression and purify the recombinant protein melittin.The protein concentration was determined,and the molecular weight of the protein expression was analyzed by SDS-PAGE electrophoresis and immune imprinting.In addition,drug sensitivity test and antibacterial test of resistant strains and toxicity test of tumor cells were carried out to determine its biological activity.The recombinant protein showed abvious destination band between 25 and 35 kDa by SDS-PAGE and grayscale analysis.The recombinant protein is highly sensitive to Staphylococcus aureus,moderately sensitive to Escherichia coli,and low sensitive to Pseudomonas aeruginosa and Shigella dysentery.Five concentrations of recombinant proteins 50,25,12.5,6.25 and 3.125 mg/L acted on MCF-7 cells,and stabilized the growth of MCF-7 cells at 12,24,36,48 and 72 h.Compared to the control group,there was a significant difference(P<0.05).Aphidopeptide fusion protein is stable and effective in prokaryotic cells via pGEX6p-1 carrier,and has obvious antibacterial and anti-tumor effects.