[1]Department of Cardiology, First Affiliated Hospital, Kunming Medical University, China.
Objectives: The purpose of this study is to establish strategies of purification of GADD45α and to detect GADD45α"s expression in human platelet and human myocardial tissue. Methods: First, the full-length coding sequence of GADD45α (498bp, GeneBank Accession No.: NM-001924.3) was amplified from a human hepatocyte cDNA library using PCR and comfirmed by sequencing. The PCR products were digested with BamH I and Xho I, and ligated into BamH I and Xho I digested pGEX-6P-1 vector with GST tag. The recombinant plasmid was transformed into E.coli BL21 (DE3). Positive clones were cultured in LB medium and induced with IPTG. The expression product was purified by affinity, molecular-exclusion and ion exchange chromatography, and then condensed to the moderate concentration. Second, the expression of GADD45α was detected in human platelet and human myocardial tissue using RT-PCR. Results: First, We established the strategy so as to clone, expression and the method of purification of GADD45α; Second, we detected GADD45α"s expression in human platelet and human myocardial tissue by RT-PCR. Conclusions: Growth arrest and DNA damage-inducible protein GADD45 alpha is an 18.3 kDa acidic nuclear protein encoded by a DNA-damage-inducible gene, also termed DDIT1, GADD45. GADD45α interacts with a number of molecules and play an important role in the regulation of DNA repair; cell cycle, cell proliferation, and apoptosis.GADD45α protein have been implicated in the development of atherosclerosis, coronary heart disease and congenital cardiovascular diseases. Research structure and function of GADD45α protein has a positive and profound significance to elucidate the pathogenesis of multiple diseases and to develop novel and effective treatment strategies. In this study, we established the strategy so as to clone, expression and the method of purification of GADD45α and determined GADD45α"s expression in human platelet and human myocardial tissue by RT-PCR. It provides experiment data for further structure and function study.
(8.0+极速模式)