Study Of Human Era Function With RNA Interference

era gene (Escherichia coli ras) was originally found as an open reading frame in E.coli. It codes a kind of GTPase, but further analysis of Era protein suggested that Era is diffrence to the large G proteins with three subunits and the small G proteins having only one structural domain . It contains an N-termainal GTP-binding domain and a conserved C-terminus that contains a putative KH domain capable of binding RNA. Up to date Era has been found widely in various organismal genome from bacterial to human and their sequences are highly conserved. Therefore, there was great significence to study this new member (Era family) of the G protein family.Era related to the cell cycle of bacteria and is very importance for bacteria survival. Deletion or mutation of era gene always leads death of the bacteria, leads great difficulties on Era function study. During professor CHEN Su-min worked in NIH, he searched the dbEST database with the C-terminal of E.coli Era and found there are Era homologues in eukaryotes, such as Caenorhabditis elegans, mouse and human. Then hecloned mouse and human era-cDNA successfully. The full length of human era-cDNA gene is 2.2 kb containings an open reading frame of 1038 bp in length that encodes a protein of 346 amino acid. The full length of mouse era-cDNA gene is 2.2 kb and contains an open reading frame of 1306 bp in length encoding a protein of 311 amino acid. Similar to bacterial Era, the eukaryotic Era also contains the N-terminus of GTPase domain and the C-terminus of KH domain. There is no report about the function of human or mouse era gene as yet. In this study, we first tried to study the function of human era gene with RNA interference.the shRNA expression vector constructed by our group, specially applied to RNAi was utilized. RNAi specially blocked the expression of hera gene in hela cell, MCF-7 cell and HHCC cell. To analyse the effect of RNA interference, We detect the quantity of Era in tumor cells with western blot .As expected, The transfected cells exhibited a much lower content of Era protein. To further validate the effect of RNA interference , we make use of indirect immunofluorescence assay. The result also demonstrated that the RNA interference is stabilization and availability.To get more information of the function of human Era, we observed a seris of experiment results, for example, flow cytometry (FCM), growth curve of cell and electron microscopy, etc. we could found that it had been slow clearly that the rate of growth of the experiment group cells.The results of FCM clew that the cell cycle had been retardarce at G2-M phase. We can observed the variety of tumor cell phenotype from whenera gene expression was come down by RNA interference. The quantity of mitochondrial increased in evidence, endoplasmic reticulum and ribosome were nearly, disappeared. These results hint Era could play a important role in the modulation of cell cycle .To obtain soluble mEra protein, the cDNA gene that encode the full length of mEra were amplified from the template of pBluescript-mera cDNA plasmid by PCR method. Then expressed the mera gene of recombinant plasmid pMSM-mera in E.coli. The expressed MBP-fused protein all existed in soluble form, and took 18% of the total bacterial protein. After purified by amylose affinity chromatography, the purity of the protein was 71%. The expressed protein (MBP-mEra) was also identified by Western blotting.