Prokaryotic Expression Of EOLA1 Gene, Purification Of Its Protein And Preparation Of Polyclonal Antibody

Sepsis is an auspice of multiple organ dysfunctional syndrome (MODS) and an intractable problem, which is facing with in critical and serious illness medicine. Sepsis is also the one of main causes of death in serious burn and traumatic patients. EC is one kind of the main target cells that are directly activated by LPS, and effect on EC by LPS with regard to signal transmitting and special receptor and route of action has been studied more. The EOLA1 gene (a human novel gene, GenBank Accession No. AY074889) was cloned in our laboratory by the effective technique of mRNA differential display (comparing the differential gene expression between normal ECV304 and treated by LPS). Using the SMART RACE technique, the full-length cDNA of the EOLA1 gene was cloned. It contains 1404 nucleotides, 474 nucleotides of the open reading frame that predicts 158 amino acids. The genomic DNA contains 5 exons, spans about 6294 bps, and is mapped to human chromosome Xq27.4. Analyzing by bioinformatics methods found that EOLA1 secondary structure contains α -helix, β -Lamellosa and β -turn, and a helix-turn-helix (HTH) motif. This information indicated that EOLAl may be as a transcription factor, which play an important role in process of activating Human EC.Northern blot with some kinds of Human tissues and carcinoma cells revealed an extended expression profile of EOLA1. It was highly expressed in skeletal muscle, liver, placenta and lowly expressed in the spleen, heart and no expressed in brain, colon, small intestine, thymus, lung and peripheral blood leukocyte; The yeast two-hybrid experiment has showed the interaction of EOLA1 and MT2A which may play some roles of apoptosis and growth and anti-inflammation in human EC. Now the biological function of EOAL1 is unknown, so preparing anti-EOLA1 antibody is very important to functional studying of EOLA1 in the future.The open reading frame of EOLA1 gene was cloned by RT-PCR from human ECV304 cells, and was judged as 500bp length by agarose gel electrophoresis. Recombinantplasmid was successfully constructed and identified by colony PCR and sequence measurement. Recombinant EOLAl protein was successfully expressed in BL21(DE3)plysS and the expressed protein in E.coli mainly exist in inclusion body form. The molecular weight of EOLAl is about 20x103 by SDS-PAGE analysing and Western blot. The optimization of induction conditions which include Incubate at 37°C for 4h, E.coli BL21(DE3)plysS, lmmol/L IPTG and 100 u g/ml ampicillin was founded through experiment. The target protein has been expressed in large-scale under above induction conditions: 9.6g of the cells collected by centrifugation from induced 2000ml culture and 0.76g of inclusion bodies were collected after primary purification including destruction of bacteria, washing-up and isolation. 100ml of target protein solution with 124.16ug/ml concentration was obtained after immobilized metal affinity chromatography and renaturation. Peptide mass fingerprinting show the matched peptides cover 32% (56/172 AA's) of the theoretic EOLAl protein. The anti-EOLAl polyclonal antibody was collected through BALB/c rat immunization and identified by Western-blot. ELISA showed its efficiency has arrived at 1:10000.In conclusion, the opening read frame of EOLAl has been cloned by RT-PCR from ECV304 cells and identified by sequence analysis, which further confirms the veracity and facticity of EOLAl. EOLAl recombinant protein can be expressed in E.coli largely in inclusion body form. Anti-EOLAl polyclonal antibody that is produced by our experiment can detect expression of nature EOLAl in ECV304 cells.