Construction of mammalian cell expression vector of human CD154 gene from active peripheral blood mononuclear cell and analysis of its sequence
10.3969/j.issn.1000-8861.2001.02.003
- VernacularTitle:人CD154基因真核细胞表达载体的构建、鉴定及序列分析
- Author:
Chunyan ZHANG
;
Bo NING
;
Shunong LI
;
Zhifang ZHANG
;
Lianqiang FENG
- Publication Type:Journal Article
- From:
Immunological Journal
2001;(2):88-90
- CountryChina
- Language:Chinese
-
Abstract:
Objective To obtain mammalian cell expression vector of human CD154 gene. Methods A 820 bp cDNA fragment was amplified by RT-PCR method from total RNA of human peripheral blood mononuclear cell（PBMC） activated with 10 ng／mL PMA and 1 μg／mL PHA for 8 hours. The fragment was cloned into pcDNA3.1（＋） plasmids.The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes BamH Ⅰ and EcoR Ⅰ and sequenced by Sangers-dideory-mediated chain termination. Results This cDNA fragment included 820 bp entire coding region and a part of the 3 non-coding region. The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was constructed， the sequence of the insert was identical to the published sequence encoding human CD154 antigen. Conclusion The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was successfully constructed.
