Study On Novel Gene Induced By IL-6

Cytokines are a series of signal transferring molecules in the immunity and hematopoietic system. All those factors perform their biological function by combination with special receptor of their target cell. An important result of cytokines' action on receptor is induction of novel gene expression.. Interleukin-6 is a sort of multifunctional cytokine, It possesses a function of accelerating growth and inducing differentiation for hematopoietic stem cells and lymphocytes, et al. When it acts on target cells, it induces the novel gene express. To clone such a gene does help to revealing mechanisms of IL-6 signal transduction and discovering new signal transduction molecules, and clarifying the relationship between IL-6 and some disease.1. Computer clone for EST correlation with IL-6, novel gene fished and bioinformatics analysisBased on the expressed sequence tag (EST) which enrolled at genebank hold hi our laboratory, computer clone by Siclone method was used to clone the full length of cDNA and they were named as E-LX1, E-LX2, E-LX3, E-LX4 and E-LX5. The alignment analysis of EST and their full length of cDNA which was acquired by Siclone had been done using the software Goldkey. With this analysis ,we found that except LX4, the other 4 EST obtained effective extension. E-LX4 only hold 44.61% concordance with the EST, so it was considered as miscarriage of justice by E-clone. Similarity search and ORF analysis for the four E-clone cDNA had been done by BLAST, and the information was obtained that LX1 and LX3 might be the cDNA that possess integral open reading frame; but unknown functional novel gene. LX4 has no encoding region, LX5 may be an intron. So we think it is meaningful to study further LX1 and LX3.The LX3 gene was analyzed for PI, Mw, Hydrophobicicy, Amino acid content, motif, domain, superfamily and ORF by bioinformatics analysis. Thefull length of LX3 is 1577bp including Kozak sequence and "AATAAA" tailing signal. The ORF from 115bp to 1038bp encodes 308 amino acids. There are Aspartic acid-rich region, EF_hand₂, Tyrosine sulfation site, Protein kinase C phosphorylation site, Casein kinase II phosphorylation site, Tyrosine kinase phosphorylation site, N-myristoylation site in its sequence.. Similarity search indicated that LX3 protein shares the highest homology with the protein named 2gbp-one member of Periplasmic binding protein, which shows that it may play an important role in the cell proliferation. From those analyses we know that LX3 has a complete open reading frame and a clarified functional cue but an unknown functional novel gene.The RT-PCR method was used to fished the novel gene LX3 from U937 cell which was induced by IL-6 by 8 hours unprecedently worldly by us. The result of DNA sequencing showed that the gene fished by RT-PCR is almost identical to the one by computer clone (with a similarity of 99.68 %).This result proved that we fished the gene successfully, while we could not fish the LX3 gene with the same method from the U937 cell which could not be induced by IL-6. This result proved preliminarily that LX3 gene is the novel gene correlated with induction by IL-6.2. Expression character analysis and cell positioning research of LX3To further analyze the function of LX3, It was characterized by RT-PCR to determine whether their expression is related to IL-6 induced or not. The Time-Expression Pattern and Concentration-Expression Pattern Analysis all confirmed that LX3 genes related to IL-6 induced..To study the express status of LX3 gene in human tissue.we synthesized full length LX3 cDNA probe tagged by 32P using AP-PCR (arbitrarily primed PCR) . The gene was assessed by the Northern Blot method using Multiple Tissue RNA Blots (MTRB). The panel was comprised of 8 human normal tissues: brain, heart, liver, lung, spleen, spermary, stomach, skeletal muscle. The result provided the evidence that LX3 was expressed by different degree in all the 8 tissue, strongly in the spleen and spermary, weakly in the brain and stomach. The ?-action control was expressed...