Expression and cloning of mCD99L2 gene from mouse B lymphoma cell line A20 and construction of its eukaryotic expression vector.
- Author:
Li-jia SHEN
1
;
Wei-yi FANG
;
Si-ming XIE
;
Ying HE
;
Hui-yong JIANG
;
Tong ZHAO
Author Information
- Publication Type:Journal Article
- MeSH: 12E7 Antigen; Animals; Antigens, CD; biosynthesis; genetics; Base Sequence; Cell Line, Tumor; Cloning, Molecular; DNA, Complementary; chemistry; genetics; Eukaryotic Cells; metabolism; Gene Expression Regulation, Neoplastic; Genetic Vectors; genetics; Lymphoma, B-Cell; genetics; pathology; Mice; Molecular Sequence Data; RNA, Messenger; biosynthesis; genetics; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA
- From: Journal of Southern Medical University 2006;26(2):144-149
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo detect and clone mCD99L2 gene from mouse B lymphoma cell line A20 and construct its eukaryotic expression vector pcDNA3.1-mCD99L2.
METHODSThe expression of mCD99L2 mRNA in A20 cell line was detected by in situ hybridization. The total RNA of A20 cells was extracted to obtain the full-length cDNA of the coding region of mCD99L2 gene by RT-PCR, the product of which was ligated into pMD18-T vector and the DNA sequence of the insert was detected. The coding regions of mCD99L2 gene was amplified from pMD-mCD99L2 by PCR using primers containing EcoR I and Xho I sites and cloned into the eukaryotic expression vector pcDNA3.1/MycHis(+).
RESULTSIn situ hybridization identified positive expression of mCD99L2 gene in the A20 cell line. The full-length cDNA of mCD99L2 coding region of A20 cell line was obtained by RT-PCR, which yielded a product of 712 bp as expected, and the DNA sequence was completely homologus to the mCD99L2 cDNA reported in GenBank. Restriction endonuclease digestion and DNA sequencing indicated that the eukaryotic expression vector pcDNA3.1(+)- mCD99L2 had been constructed successfully.
CONCLUSIONmCD99L2 cDNA has been cloned from mouse B lymphoma cell line A20 and its eukaryotic expression vector pcDNA3.1(+)- mCD99L2 successfully constructed, which facilitates further functional study of mCD99L2 gene in mouse B lymphoma cell line A20.
