PIAS3的Myc融合蛋白真核表达重组质粒的构建

doi:国家自然科学基金青年科学基金项目资助课题

PIAS3的Myc融合蛋白真核表达重组质粒的构建

李江¹，甄园丽²，杨南扬²，张俊芳²，王中南²，李晓萌²

(1.吉林大学口腔医院修复科，吉林长春 130041； 2.东北师范大学生命科学学院，吉林长春 130024)

收稿日期:2008-09-19 修回日期:1900-01-01 出版日期:2009-03-28 发布日期:2009-08-14
通讯作者: 李江

Construction of eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein

LI Jiang¹， ZHEN Yuan-li²， YANG Nan-yang²， ZHANG Jun-fang,WANG Zhong-nan²， LI Xiao-meng²

(1.Department of Prosthodontics,Stomatology Hospital,Jilin University，Changchun 130041,China；2.School of Life Sciences,Northeast Normal University,Changchun 130024,China)

Received:2008-09-19 Revised:1900-01-01 Online:2009-03-28 Published:2009-08-14
Contact: LI Jiang

摘要/Abstract

摘要： 目的：构建PIAS3 （活化型STAT3抑制蛋白）的Myc融合蛋白真核表达重组质粒，并表达融合蛋白Myc-PIAS3。方法：采用PCR方法，扩增鼠PIAS3基因全长cDNA编码区序列。将该1 851 bp的特异性片段连接到T载体上，将其亚克隆至pCMV-Myc真核表达载体的SalⅠ和NotⅠ位点之间。将pCMV-Myc-PIAS3重组质粒转染前列腺癌PC3细胞，利用Myc抗体通过Western blotting法检测融合蛋白Myc-PIAS3的表达。结果：重组pCMV-Myc-PIAS3质粒通过酶切和测序鉴定了其正确性。EcoRⅠ酶切鉴定时有4 357和1 318 bp 2条带，XbaⅠ酶切鉴定时有3 291 bp和2 384 bp 2条带，与预期结果一致。测序结果显示，13个氨基酸Myc在N端，然后是阅读框架正确的PIAS3的基因序列。pCMV-Myc-PIAS3重组质粒转染PC3细胞，利用Myc抗体通过Western blotting法检测融合蛋白Myc-PIAS3的表达，在相对分子质量68 000处检测到特异的蛋白表达条带。结论：成功构建pCMV-Myc-PIAS3重组质粒，并表达融合蛋白Myc-PIAS3。

关键词: Myc融合蛋白, 重组质粒

Abstract: Abstract:Objective To construct the eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein and express the fusion protein Myc-PIAS3.Methods The full length PIAS3 fragment of 1 851bp was amplified by PCR and ligated into pMD18-T vector. The full length PIAS3 fragment was subcloned into eukaryotic pCMV-Myc vector between SalⅠ and NotⅠ sites.The recombinant pCMV-Myc-PIAS3 plasmid was trandfected into PC3 cells.The eukaryotic expression Myc-PIAS3 protein was checked by Western blotting with Myc antibody.Results The recombinant plasmid showed right sequence by the full length sequencing.The recombinant plasmid of pCMV-Myc-PIAS3 was identified by enzyme digestion.As expected,by EcoRⅠ digestion,it showed two bands of 4 357bp and 1 318 bp. By XbaⅠdigestion,it showed two bands of 3 291 bp and 2 384 bp.The sequencing result showed a N-terminal Myc of 13 amino acids followed with PIAS3 gene sequence in right reading frame.The pCMV-Myc-PIAS3 plasmid was transfected into prostate cancer PC3 cells.A specific protein expression band at relative molecular mass 68 000 was obtained by using Myc-antibody with Western blotting method.Conclusion The recombinant plasmid of pCMV-Myc-PIAS3 is sucssesefully constructed,and Myc-PIAS3 fusion protein is sucssesefully expressed.

Key words: Myc fusion protein, recombinant plasmid

中图分类号:

李江，甄园丽，杨南扬，张俊芳，王中南，李晓萌. PIAS3的Myc融合蛋白真核表达重组质粒的构建[J]. J4, 2009, 35(2): 201-204.

LI Jiang， ZHEN Yuan-li， YANG Nan-yang， ZHANG Jun-fang,WANG Zhong-nan， LI Xiao-meng. Construction of eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein[J]. J4, 2009, 35(2): 201-204.

[1]	胡志宏, 赵大力, 谢忠伟, 王梦莹, 胡景坤, 龚守良, 刘扬, 齐亚莉. pEgr1-TRAIL重组质粒联合电离辐射对乳腺癌MCF-7细胞死亡受体通路相关基因和蛋白表达的影响[J]. 吉林大学学报(医学版), 2016, 42(03): 419-423.
[2]	刘莉, 张志民, 王丽颖, 郑鹏, 李会. 变形链球菌耐氟菌株中耐酸相关基因gluA 突变的检测及其意义[J]. J4, 2011, 37(1): 80-83.
[3]	孙江滨, 谢众上, 赵科研, 李博, 柳克祥. 重组质粒Psilencer1.0-U6-siRNA-stat3对移植静脉新生内膜的增生抑制作用[J]. J4, 2010, 36(2): 299-302.
[4]	吴扬, 李玉琴, 王智昊, 臧秀贤. 人纤溶酶真核表达重组质粒的构建[J]. J4, 2009, 35(5): 898-901.
[5]	李江, 张俊芳, 甄园丽, 杨南扬, 李晓萌. pCMV-Myc-PIASxβ重组质粒的构建与蛋白表达[J]. J4, 2009, 35(3): 415-418.
[6]	董丽华，刘锋，李艳博，龚守良. 体外稳定转染的重组质粒pEgr-hp53联合辐射对人卵巢癌SKOV-3 细胞周期进程及增殖的影响[J]. J4, 2008, 34(3): 357-360.