金葡菌类肠毒素K原核表达载体构建及其生物学活性分析

doi:10.13523/j.cb.20151207

中国生物工程杂志

2015, Vol. 35

Issue (12): 45-50 DOI: 10.13523/j.cb.20151207

研究报告

金葡菌类肠毒素K原核表达载体构建及其生物学活性分析

刘彦礼^1,2, 牛荣成¹, 杨芬¹, 朱文慧¹, 林俊堂^1,2

1. 新乡医学院生命科学技术学院新乡 453003;
2. 河南省医用组织再生重点实验室新乡 453003

The Construction and Functional Analysis of Staphylococcal Enterotoxin-like K

LIU Yan-li^1,2, NIU Rong-cheng¹, YANG Fen¹, ZHU Wen-hui¹, LIN Jun-tang^1,2

1. College of Life Science and Technology, Xinxiang Medical University, Xinxiang 453003, China;
2. Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang 453003, China

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摘要：

目的:金葡菌类肠毒素K(SElK)原核表达载体构建及其生物学活性初步分析。方法:通过常规分子生物学技术将SElK基因片段插入载体pET-28a中,阳性单菌落扩大培养,诱导目的基因表达后通过Ni⁺亲和层析纯化SElK重组蛋白;小鼠脾淋巴细胞增殖实验及尾静脉注射后血常规检测SElK超抗原活性;进一步通过检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)和肌酸激酶(CK)活性及尿素(Urea)和肌酐(CR)含量来评价SElK潜在肠毒性。结果:成功构建SElK原核表达载体,并通过Ni⁺亲和层析获得高纯度SElK重组蛋白(>95%);随后的超抗原活性检测发现SElK能够在体外刺激小鼠脾淋巴细胞显著增殖,尾静脉注射后能够有效地刺激血液中淋巴细胞增殖;最终的肠毒性检测结果发现SElK能够显著增加血清中AST活性及Urea含量。结论:成功构建并纯化获得高纯度SElK重组蛋白,并对其超抗原活性及潜在肠毒性进行初步分析,为开发更优的抗肿瘤制剂提供备选药物。

关键词： 超抗原; T淋巴细胞; 肠毒性; 金葡菌类肠毒素; 血常规

Abstract:

Objective:The aim is to construct the prokaryotic expression vector of Staphylococcal Enterotoxin-like K (SElK), and preliminarily determine its' biological activity. Methods:Firstly, the target gene SElK was cloned into the plasmid of pET-28a, after being confirmed by colony PCR, double digestion and sequence, the purified recombined protein SElK was achieved by Ni⁺-affinity chromatography, and examined by SDS-PAGE. Subsequently, the superantigen activity of SElK was examined by stimulating the proliferation of spleen lymphocytes in vitro, and the routine blood tests in vivo after SElK injection from tail vein. Finally, the intrinsic enterotoxigenesis of SElK was evaluated by examined the activity or content of key organs' important indicators in the serum after SElK injection from tail vein, including ALT, AST, LDH, CK, Urea and CR. Results:Prokaryotic expression vector of pET-28a-SElK was successfully constructed and high purity (>95%) recombined protein SElK was achieved by Ni⁺-affinity chromatography; the subsequent analysis of superantigen activity showed that SElK could significantly stimulate the proliferation of spleen lymphocytes in vitro, and the further routine blood tests also demonstrated that SElK could induce the expension of lymphocytes in vivo; the final enterotoxigenesis tests showed that SElK could significantly increase the activity of AST and the content of Urea. Conclusion:The provided fundamental data for the characteristics of SElK, and the results suggested that SElK could be used as a potent candidate agent for tumour treatment in clinics.

Key words: Routine blood test Enterotoxigenesis Staphylococcal Enterotoxin-like Superantigen T cells

收稿日期: 2015-10-13 出版日期: 2015-12-22

ZTFLH:

Q819

基金资助:

国家自然科学基金(31502045)资助项目

通讯作者: 林俊堂 E-mail: linjtlin@126.com

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引用本文:

刘彦礼, 牛荣成, 杨芬, 朱文慧, 林俊堂. 金葡菌类肠毒素K原核表达载体构建及其生物学活性分析[J]. 中国生物工程杂志, 2015, 35(12): 45-50.

LIU Yan-li, NIU Rong-cheng, YANG Fen, ZHU Wen-hui, LIN Jun-tang. The Construction and Functional Analysis of Staphylococcal Enterotoxin-like K. China Biotechnology, 2015, 35(12): 45-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151207 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I12/45

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