EMMPRIN磷酸化突变质粒的构建及功能检测

doi:10.7503/cjcu20140141

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (10): 2104.doi: 10.7503/cjcu20140141

EMMPRIN磷酸化突变质粒的构建及功能检测

赵超悦¹, 王娜², 王娟婧², 贺玺¹, 李江¹()

1. 吉林大学口腔医院修复科, 长春 130021
2. 东北师范大学生命科学学院, 长春 130024

收稿日期:2014-02-25 出版日期:2014-10-10 发布日期:2014-09-30
作者简介:联系人简介: 李江, 男, 博士, 教授, 主要从事口腔修复学临床与基础研究. E-mail: ljiang@jlu.edu.cn
基金资助:
吉林省科技发展计划基金(批准号: YYZX201241)、吉林省自然科学基金(批准号: 201115106)和长春市科技局项目(批准号: 2011122)资助

Construction and Functional Detection of Mutant Plasmid EMMPRIN Phosphorylation^†

ZHAO Chaoyue¹, WANG Na², WANG Juanjing², HE Xi¹, LI Jiang^1,^*()

1. Department of Prothodontics, Stomatology Hospital, Jilin University, Changchun 130021, China
2. School of Life Sciences, Northeast Normal University, Changchun 130024, China

Received:2014-02-25 Online:2014-10-10 Published:2014-09-30
Contact: LI Jiang E-mail:ljiang@jlu.edu.cn
Supported by:
Supported by the Science and Technology Development Fund of Jilin Province, China(No.YYZX201241), the Natural Science Foundation of Jilin Province, China(No.201115106) and Changchun Municipal Science and Technology Bureau, China(No.2011122)

摘要/Abstract

摘要：

利用聚合酶链式反应(PCR)定点诱变技术, 对细胞外基质金属蛋白酶诱导因子(EMMPRIN)的2个磷酸化位点进行突变, 构建了EMMPRIN磷酸化位点突变质粒. 对其功能进行检测发现, EMMPRIN磷酸化位点突变质粒可在细胞内正常表达. 研究结果显示, 突变质粒可抑制肿瘤细胞的增殖(P<0.05), 表明EMMPRIN可能与肿瘤细胞的增殖相关.

关键词: 细胞外基质金属蛋白酶诱导因子(EMMPRIN), 点突变, 磷酸化

Abstract:

In order to investigate whether the proliferation of tumor cells associated with the mutant EMMPRIN phosphorylation site plasmid constructed by mutating the 246th, the 252th serine to alanine in the sequence of extracellular matrix metalloproteinase inducer, two extracellular matrix metalloproteinase inducer(EMMPRIN) phosphorylation sites were found out and mutated to construct the mutant EMMPRIN phosphorylation site plasmid with PCR site-directed mutagenesis techniques, and then to demonstrate the plasmid could be normally expressed within the cell through functional detection. The results revealed that the mutant-type EMMPRIN inhibits the proliferation of tumor cells(*P<0.05), which demonstrate that EMMPRIN may be associated with the proliferation of tumor cells.

Key words: Extracellular matrix metalloproteinase inducer(EMMPRIN), Point mutation, Phosphorylation(Ed.: P, H, N, K)

中图分类号:

O629.7

TrendMD:

赵超悦, 王娜, 王娟婧, 贺玺, 李江. EMMPRIN磷酸化突变质粒的构建及功能检测. 高等学校化学学报, 2014, 35(10): 2104.

ZHAO Chaoyue, WANG Na, WANG Juanjing, HE Xi, LI Jiang. Construction and Functional Detection of Mutant Plasmid EMMPRIN Phosphorylation^†. Chem. J. Chinese Universities, 2014, 35(10): 2104.

图/表 3

Table 1 Primers for phosphorylation mutation

Primer name	Primer sequence(5'—3')	Primer name	Primer sequence(5'—3')
S246A Forward	GCCGGCGCTGCACCCCTGAAGAGC	S252A Forward	AAGAGCGCCGGGCAGCACCAGAAT
S246A Reverse	GGGTGCAGCGCCGGCGTCGTCATC	S252A Reverse	CTGCCCGGCGCTCTTCAGGGGTGC

Fig.1 EMMPRIN mutant cell morphology(arrow referred to as filopodia) EMMPRIN/GFP is a plasmid which carried the green fluorescent protein, and observed under green fluorescence microscope. Phalloidin combined with the F-actin, which showed that the distribution of microfilament skeleton in cells, and observed under red fluorescence microscope. 293T cells were treated with 1 μg EMMPRIN plasmid or 1 μg EMMPRIN(S246A) or 1 μg EMMPRIN(S252A) plasmid.(Arrows indicate filopodia).

Fig.2 Impact of wild-type and mutant-type EMMPRIN at different time periods on cell proliferation 293T cells were treated with wild-type and mutant-type EMMPRIN at various times. Survival rates of cells were determined by MTT assay.Results are presented as means±SD(error bars) of triplicate experiments and are expressed as a percentage of untreated control cells, *P<0.05 compared with group of wild-type.

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EMMPRIN磷酸化突变质粒的构建及功能检测

Construction and Functional Detection of Mutant Plasmid EMMPRIN Phosphorylation^†

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EMMPRIN磷酸化突变质粒的构建及功能检测

Construction and Functional Detection of Mutant Plasmid EMMPRIN Phosphorylation†

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Construction and Functional Detection of Mutant Plasmid EMMPRIN Phosphorylation^†