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 <sup>*</sup>

doi:10.13523/j.cb.20181203

中国生物工程杂志

2018, Vol. 38

Issue (12): 14-20 DOI: 10.13523/j.cb.20181203

研究报告

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 ^*

何亚南^1,²,孙钰椋^1,²,任雅坤²,梁盛英²,杨芬^2,³,刘彦礼^1,^2,^**(

),林俊堂^1,^2,³

1 新乡医学院生命科学技术学院新乡 453003
2 新乡医学院河南省医用组织再生重点实验室新乡 453003
3 新乡医学院生物医学工程学院新乡 453003

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

HE Ya-nan^1,²,SUN Yu-liang^1,²,REN Ya-kun²,LIANG Sheng-ying²,YANG Fen^2,³,LIU Yan-li^1,^2,^**(

),LIN Jun-tang^1,^2,³

1 College of Life Science and Technology,Xinxiang Medical University, Xinxiang 453003,China
2 Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang Medical University, Xinxiang 453003,China
3 College of Biomedical Engineering, Xinxiang Medical University, Xinxiang 453003,China

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摘要：

目的构建携带金黄色葡萄球菌类肠毒素K(staphylococcal enterotoxin-like K, 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Overlap PCR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.coli BL21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。

关键词： 金黄色葡萄球菌类肠毒素K; 超抗原; T淋巴细胞; 原核表达

Abstract:

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.

Key words: Staphylococcal enterotoxin-like K Superantigen T lymphocyte Prokaryotic expression

收稿日期: 2018-06-06 出版日期: 2019-01-10

ZTFLH:

Q78

基金资助: * 国家自然科学基金(31502045)

通讯作者: 刘彦礼 E-mail: liuyanli198512@163.com

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引用本文:

何亚南,孙钰椋,任雅坤,梁盛英,杨芬,刘彦礼,林俊堂. 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 ^*[J]. 中国生物工程杂志, 2018, 38(12): 14-20.

HE Ya-nan,SUN Yu-liang,REN Ya-kun,LIANG Sheng-ying,YANG Fen,LIU Yan-li,LIN Jun-tang. The Construction and Functional Analysis of Staphylococcal Enterotoxin-like K and GFP Fusion Protein. China Biotechnology, 2018, 38(12): 14-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181203 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I12/14

表1 SElK、GFP和SElK-GFP基因克隆所需PCR引物

图1 SElK-GFP原核表达载体构建及鉴定

图2 SElK-GFP的诱导表达过程中绿色荧光的检测

图3 SElK-GFP蛋白的纯化和体外刺激小鼠淋巴细胞增殖活性检测

图4 SElK-GFP体内刺激细胞因子IL-2、IFN-γ的分泌水平

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