金黄色葡萄球菌类肠毒素K与GFP融合蛋白工程菌的构建及其表达蛋白生物学活性分析

The Construction and Functional Analysis of Staphylococcal Enterotoxin-like K and GFP Fusion Protein

目的构建携带金黄色葡萄球菌类肠毒素K(staphylococcal enterotoxin-likeK,SElK)和绿色荧光蛋白(green fluorescent protein,GFP)融合基因的工程菌,并对SElK-GFP融合蛋白进行初步生物学活性分析。方法利用PCR和OverlapPCR克隆获得SElK-GFP融合基因,并插入pET28a表达载体中,通过菌落PCR,质粒双酶切及测序验证后,将构建成功的pET28a-SElK-GFP质粒转化到E.coliBL21菌株中进行诱导表达,通过Ni+亲和磁珠试剂盒纯化获得SElK-GFP融合蛋白;并利用MTT法检测SElK-GFP刺激小鼠脾淋巴细胞增殖;ELISA法检测SElK-GFP尾静脉注射后小鼠血清中细胞因子IL-2和IFN-γ的分泌水平。结果成功构建能够表达SElK-GFP融合蛋白的工程菌,纯化获得高纯度的SElK-GFP融合蛋白可观测到明显的绿色荧光,融合蛋白生物学活性分析表明,SElK-GFP能够呈剂量依赖性地显著刺激小鼠脾淋巴细胞增殖;同时ELISA检测发现SElK-GFP可显著增加小鼠血清中细胞因子IL-2及IFN-γ的分泌水平。结论成功克隆、表达及纯化获得高纯度的SElK-GFP融合蛋白,其不仅保留了SElK的超抗原活性,同时兼具GFP绿色荧光的可视性,为深入研究SElK生物学活性提供有利工具。

Objective:The aim of this study is to construct the genetically engineered bacteria with full-length fusion gene of staphylococcal enterotoxin-like K (SElK) and green fluorescent protein (GFP),and further examine the biological activity of SElK-GFP fusion protein.Methods:SElK-GFP fusion gene was obtained by overlap PCR and cloned into the plasmid of pET28a.After being confirmed by colony PCR,double digestion and sequence,the successfully constructed pET28a-SElK-GFP was transformed into E.coli BL21,and the SElK-GFP was purified by Ni +-affinity magnetic beads.The proliferation of mouse derived spleen lymphocytes stimulated with SElK-GFP was examined by MTT assay; the concentration of IL-2 and IFN-γ in serum of the mice treated with SElK-GFP was examined by ELISA kits.Results:The SElK-GFP-producing strain was successfully constructed,and the green fluorescence can be observed in high purity SElK-GFP fusion protein.MTT assay showed that SElK-GFP could significantly stimulate the proliferation of spleen lymphocytes in a dose-dependent manner,and the concentration of IL-2 and IFN-γ in serum of the mice treated with SElK-GFP was significantly increased.Conclusion:SElK-GFP not only retained the green fluorescence signal of GFP,but also exhibited SElK superantgen activity,and provide a promising tool for the further study of the biological activity of SElK.