Article(id=1242132975555518663, tenantId=1146029695717560320, journalId=1146031591421210625, issueId=1242132974196564162, articleNumber=null, orderNo=3, doi=10.3981/j.issn.1000-7857.2015.01.002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1401984000000, receivedDateStr=2014-06-06, revisedDate=1412006400000, revisedDateStr=2014-09-30, acceptedDate=null, acceptedDateStr=null, onlineDate=1422861959993, onlineDateStr=2015-02-02, pubDate=1421078400000, pubDateStr=2015-01-13, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1422861959993, onlineIssueDateStr=2015-02-02, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1774077179083, creator=sys-migrate, updateTime=1774077179083, updator=sys-migrate, issue=Issue{id=1242132974196564162, tenantId=1146029695717560320, journalId=1146031591421210625, year='2015', volume='33', issue='1', pageStart='1', pageEnd='128', issueExtLink='null', onlineDate='null', pubDate='1421078400000', pubDateStr='2015-01-13', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=3, issueType=-1, specialIssue=null, createTime=1774077178760, creator='sys-migrate', updateTime=1774077178760, updator='sys-migrate', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=17, endPage=21, ext={EN=ArticleExt(id=1242132977862390115, articleId=1242132975555518663, tenantId=1146029695717560320, journalId=1146031591421210625, language=EN, title=Detecting expression of spindle protein gene INMAP in hepatoma cells by polyclonal antibody, columnId=null, journalTitle=Science & Technology Review, columnName=null, runingTitle=null, highlight=null, articleAbstract=To investigate the function of INMAP of spindle protein and its role in malignant cell proliferation, an INMAP polyclonal antibody is prepared. The prokaryotic expression of His-INMAP fusion protein is induced at 16℃ by adding 0.1 mmol/L isopropyl-β -D-thiogalactoside (IPTG), which is identified by SDS-PAGE analysis and western blotting assay. The inclusion body is treated with 4.0 mol/L urea to obtain soluble His-INMAP fusion protein antibody, fusion protein is purified using Ni Sepharose High Performance, and then the protein concentration and purity are detected. The purified protein antibody is injected into 4 Balb/c mice, then blood samples are collected from their hearts, and the anti- serum is isolated. The specificity of polyclonal anti- INMAP antibodies in unpurified and purified prokaryotically expressed products are analysed. In addition, the expression difference between normal liver cell L-02 and 5 hepatoma cell (PLC, HepG2, SUN449, SMMC-7721 and BEL-7402) is determined by western blotting assay. The results show that His-INMAP fusion protein mainly exists in insoluble inclusion bodies. Soluble protein is obtained with 4.0 mol/L urea treatment to solubilise inclusion bodies. Highly protein purity (94.1%) is harvested after purification. The polyclonal anti-INMAP antibody can bind antigen specifically. Moreover, INMAP is found existing polymorphically in hepatoma cells and its gene expression is down-regulated significantly in all tested hepatoma cells except PLC cell. Obviously, in this study the anti-INMAP polyclonal antibody is of high specificity, which lays a foundation of further study of INMAP functions., authors=ZHU Yan1,2 , KANG Jingyan1,4 , WANG Yu1 , SUN Le3 , ZHANG Haijiang3 , LIANG Qianjin1,2 , authorsList=ZHU Yan, KANG Jingyan, WANG Yu, SUN Le, ZHANG Haijiang, LIANG Qianjin, authorCompany=1. Beijing Key Laboratory of Gene Resource and Molecular Development, School of Life Sciences, Beijing Normal University, Beijing 100875, China;INMAP 基因功能的进一步研究奠定了基础., authors=朱艳1,2 , 康璟妍1,4 , 王钰1 , 孙乐3 , 张海江3 , 梁前进1,2 , authorsList=朱艳, 康璟妍, 王钰, 孙乐, 张海江, 梁前进, authorCompany=1. 北京师范大学生命科学学院, 抗性基因资源与分子发育北京市重点实验室, 北京100875;
科技导报
| 研究论文 2015, 33(1): 17-21
利用多克隆抗体检测纺锤体蛋白基因INMAP在肝癌细胞中的表达
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朱艳1,2 , 康璟妍1,4 , 王钰1 , 孙乐3 , 张海江3 , 梁前进1,2
作者信息
1. 北京师范大学生命科学学院, 抗性基因资源与分子发育北京市重点实验室, 北京100875;
通讯作者:
梁前进(通信作者),教授,研究方向为分子细胞遗传与功能基因,电子信箱:Lqj@bnu.edu.cn
Detecting expression of spindle protein gene INMAP in hepatoma cells by polyclonal antibody
Affiliations
出版时间: 2015-01-13
doi: 10.3981/j.issn.1000-7857.2015.01.002
文章导航
为探究纺锤体蛋白INMAP 的功能及其在细胞恶性增殖中发挥的作用,制备INMAP 多克隆抗体.利用0.1 mmol/L IPTG于16℃诱导His-INMAP 融合蛋白进行原核表达,SDS-PAGE 及免疫印迹检测蛋白表达.4.0 mol/L 尿素处理包涵体获得可溶性融合蛋白,镍亲和柱分离纯化融合蛋白抗原并检测蛋白浓度及纯度.以所获抗原免疫4 只Balb/c 小鼠,收集心脏抗血清.以纯化前、后的原核表达产物作为抗原,检测INMAP 多克隆抗体的特异性.通过免疫印迹分析INMAP 在正常肝细胞系L-02 及5个肝癌细胞系(PLC、HepG2、SUN449、SMMC-7721 和BEL-7402)中的表达差异.结果显示,His-INMAP 融合蛋白主要以包涵体形式存在,经4.0 mol/L 尿素处理可获得可溶性目的蛋白,且纯化后的抗原纯度较高(94.1%).INMAP 多克隆抗体能与INMAP 抗原特异结合.INMAP 在6 种肝细胞中具有多态性,除PLC 外,在其他肝癌细胞中其基因表达水平显著下调.本研究制备的INMAP 多克隆抗体特异性高,为INMAP 基因功能的进一步研究奠定了基础.
INMAP
/
肝癌细胞
/
多克隆抗体
/
包涵体
To investigate the function of INMAP of spindle protein and its role in malignant cell proliferation, an INMAP polyclonal antibody is prepared. The prokaryotic expression of His-INMAP fusion protein is induced at 16℃ by adding 0.1 mmol/L isopropyl-β -D-thiogalactoside (IPTG), which is identified by SDS-PAGE analysis and western blotting assay. The inclusion body is treated with 4.0 mol/L urea to obtain soluble His-INMAP fusion protein antibody, fusion protein is purified using Ni Sepharose High Performance, and then the protein concentration and purity are detected. The purified protein antibody is injected into 4 Balb/c mice, then blood samples are collected from their hearts, and the anti- serum is isolated. The specificity of polyclonal anti- INMAP antibodies in unpurified and purified prokaryotically expressed products are analysed. In addition, the expression difference between normal liver cell L-02 and 5 hepatoma cell (PLC, HepG2, SUN449, SMMC-7721 and BEL-7402) is determined by western blotting assay. The results show that His-INMAP fusion protein mainly exists in insoluble inclusion bodies. Soluble protein is obtained with 4.0 mol/L urea treatment to solubilise inclusion bodies. Highly protein purity (94.1%) is harvested after purification. The polyclonal anti-INMAP antibody can bind antigen specifically. Moreover, INMAP is found existing polymorphically in hepatoma cells and its gene expression is down-regulated significantly in all tested hepatoma cells except PLC cell. Obviously, in this study the anti-INMAP polyclonal antibody is of high specificity, which lays a foundation of further study of INMAP functions.
INMAP
/
hepatoma (liver cancer) cell
/
polyclonal antibody
/
inclusion body
朱艳, 康璟妍, 王钰, 孙乐, 张海江, 梁前进.
利用多克隆抗体检测纺锤体蛋白基因INMAP在肝癌细胞中的表达.
科技导报,
2015
, 33
(1)
: 17
-21
.
DOI: 10.3981/j.issn.1000-7857.2015.01.002
ZHU Yan, KANG Jingyan, WANG Yu, SUN Le, ZHANG Haijiang, LIANG Qianjin.
Detecting expression of spindle protein gene INMAP in hepatoma cells by polyclonal antibody[J].
Science & Technology Review ,
2015
, 33
(1)
: 17
-21
.
DOI: 10.3981/j.issn.1000-7857.2015.01.002
2015年第33卷第1期
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文章信息
doi: 10.3981/j.issn.1000-7857.2015.01.002
接收时间：2014-06-06
首发时间：2015-02-02
出版时间：2015-01-13
收稿日期：2014-06-06
修回日期：2014-09-30
通讯作者:
梁前进(通信作者),教授,研究方向为分子细胞遗传与功能基因,电子信箱:Lqj@bnu.edu.cn
https://castjournals.cast.org.cn/joweb/kjdb/CN/10.3981/j.issn.1000-7857.2015.01.002
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2种不同金属材料的力学参数
科 属数 种数 占总种数比例 属 种数 占总种数比例 鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78 小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39 多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39 红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87 小菇属 Mycena 11 5.26 光柄菇属 Pluteus 5 2.39 红菇属 Russula 17 8.13 栓菌属 Trametes 5 2.39
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