Cloning And Expression Of Human DJ-1

Objective Parkinson's disease (PD) is a common neurodegenerative disorder pathologically by loss of dopaminergic neurons and accumulation of eosinophilic inclusions termed Lewy bodies (LBs) in cells of the substantia nigra pars compacta(SNpc), andα-Synuclein is the major component of Lewy bodies (LBs). So far, the mechanisms underlying the pathological effects of PD remain unclear, but it is agreed that gene and environmental influences which affect each other may induce PD. Recent studies found that certain mutations in DJ-1 are related to autosomal-dominant early onset familial Parkinson's disease (PD) and other forms of DJ-1 mutation have been found in some sporadic PD patients. It has been reported many times that DJ-1 might modulate transform, resist oxidative stress, and may be a molecular chaperon. At the same time, dysfunction of mitochondria under oxidative stress and abnormal accumulation of certain protein are considered to play a very important role in PD. These studies suggest that DJ-1 may be implicated in the pathogenesis of PD, the dysfunction resulted from mutations in DJ-1 might lead to the loss of dopaminergic neurons and the normal DJ-1 which can resist oxidative stress or function as a molecular chaperon might be a protective protein. However, the mechanisms underlying its pathological effects remain unclear. The purpose of present study therefore, is to clone the cDNA of human DJ-1 from Chinese fetal liver and to construct the expression vector, pcDNA3.1-DJ-1, which could express human wild-type DJ-1 in eukaryotic cells. This study will help us further understand the functions of DJ-1 as well the relationship between DJ-1 and the pathogenesis of PD. Moreover, do some contribution to the treatment of PD.Method In order to clone the human gene of DJ-1, total cellular RNA was isolated from human fetal liver and reversely transcribed into cDNA, which was amplified by PCR (polymerase chain reaction) using 5'-AAGCTTGGGTGCAGGCTTGTAAAC-3' and 5'-TCTAGAAGTGATCGTCGCAGTTCG-3' as the specific primers. The resultant PCR product was cloned into pGEM-T Easy vector for sequencing after enzymatic analysis with HindⅢand XbaI. After DNA sequence analysis which showed that the full long cDNA of DJ-1 was obtained and was identical to that in the GeneBank, DJ-1 cDNA was subcoloned into pcDNA3.1 vector to create eukaryotic expression vector pcDNA3.1-DJ-1. The recombinant vector pcDNA3.1-DJ-1 was digested with HindⅢand XbaI and then was identified through agarose gel electrophoresis analogy.In order to detect the expression of pcDNA3.1-DJ-1 in human eukaryotic cells, the human dopaminegergic cell line SH-SY5Y was transfected by the plasmid pcDNA3.1 with LipofectAMINE. Negative control group is untransfected SH-SY5Y cell. The expression of DJ-1 in transfected cells was determined by RT-PCR, western blot, and measured cell activity using MTT assay.Results The resultant RT-PCR product was detected by 1% agarose gel electrophoresis and was shown to have a specific fragment of 636bp. Insert the PCR product into pGEM-T Easy vector, the DNA sequencing showed that the full long cDNA of DJ-1 was obtained which was identical to that in the GenBank. Subcoloned DJ-1 cDNA into pcDNA3.1 vector to create eukaryotic expression vector pcDNA3.1-DJ-1. The analysis of restriction fragment from 1 % agarose gel elctrophoresis showed that the recombinant vector, pcDNA3.1-DJ-1, was composed by two pieces, namely DJ-1 and pcDNA3.1. These rusults suggested that pcDNA3.1-DJ-1 vector was constructed successfully.The expression of pcDNA3.1-DJ-1 in transfected cells was determined by RT-PCR and western blot. The result of RT-PCR showed that there was a 636bp DNA band as expected in transfected SH-SY5Y cells, while there was not any DNA band in untransfected SH-SY5Y cells. A 20kD protein from the transfected SH-SY5Y cells could be observed by western blot that is expected size of DJ-1, so do the untransfected SH-SY5Y cells. But the protein band from the transfected SH-SY5Y cells was lighter than that from the untransfected SH-SY5Y cells. Detect the growth curve of SH-SY5Y cells and transfected SH-SY5Y cells through MTT, the result showed that both kinds of cells indicated good state and the activity of the transfected cells was equal to that of the SH-SY5Y cells.Conclusion Clone the Chinese DJ-1 gene successfully and construct the recombinant vector pcDNA3.1-DJ-1 which can express in SH-SY5Y cells successfully.