Construction of eukaryotic expression plasmid pEGFP-N3-M-IL-2(88 Arg,125 Ala) and its effects on Glioma cell line U87
10.3969/j.issn.1000-484X.2014.12.013
- VernacularTitle:真核表达载体 pEGFP-N3-M-IL-2（88 Arg，125 Ala）的构建表达及对神经胶质瘤细胞U87的影响
- Author:
Guangcan SHAO
;
Dongmeng QIAN
;
Ming HU
;
Lina ZHANG
;
Xuxia SONG
;
Jiazhen FU
;
Bin WANG
- Publication Type:Journal Article
- Keywords:
Interleukin-2;
Melittin;
Eukaryotic expression;
Glioma cells
- From:
Chinese Journal of Immunology
2014;(12):1647-1651,1657
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a recombinant eukaryotic expressive vector of pEGFP-N3-M-IL-2(88 Arg,125 Ala),and to study the expression of this gene in the Glioma cell line U 87,and to detect its antitumor activities of the fusion protein M-IL-2(88 Arg,125 Ala).Methods:The target fusion gene M-IL-2 (88 Arg,125 Ala) was amplified by PCR from pPICZαA/M-IL-2 (88 Arg,125 Ala) and cloned into pEGFP-N3 vector after digestion to construct recombinant eukaryotic expressive vector pEGFP -N3-M-IL-2( 88 Arg,125 Ala).And then recombinant plasmid pEGFP-N3-M-IL-2(88Arg,125Ala) was transfected into Glioma cell U87 by LipofectamineTM2000 immediately after it was confirmed by restrictive enzyme analysis and sequencing .RT-PCR and Western blot were used to confirm expression of the fusion gene in the U87.Prohibitory effect of recombinant M-IL-2(88Arg,125Ala) protein on U87 was assessed by CCK-8 assay.Results:Restrictive analysis and sequence analysis revealed that M-IL-2(88Arg,125Ala) fusion gene was cloned into the vector pEGFP-N3 suc-cessfully,fusion gene M-IL-2(88Arg,125Ala) could express in U87 cells and could inhibit the growth of U87 cells.Conclusion:The eu-karyotic expression plasmid pEGFP-N3-M-IL-2( 88 Arg,125 Ala) was constructed and expressed in U 87 cells successfully ,the fusion protein could inhibit the growth of U87 cells.We laid a foundation for further research of gene M-IL-2(88 Arg,125 Ala).
