Cloning, Expression ,Purification And Bioinformatics Analysis Of Genes Related With IL-6

Our known is limited for IL-6 signal transduction pathway and corresponding signal molecule. The isolation of genes related to IL-6 is necessary for revealing mechanisms of IL-6 signal transduction and discovery of new signal transduction molecules. To clone genes related with IL-6, here we utilized cDNA-RDA and a unproved DD-PCR method to both detect and isolate cDNA fragments from differentially expressed genes from the IL-6 induced and uninduced U937 cells. By using cDNA-Representational Differences Anailysis(RDA) and DD-PCR, we found seven gene fragments which include some known genes such as Human chromodomain helicase-DNA-binding protein 4 (CHD-4) and homology to homeobox protein DLX-2 that formerly suggested to play a role in cell life and signal transduction. Nine genes were verified completely novel after searching the dbEST and SwissProt data base in GenBank and were submitted to GenBank. The accession numbers for them are, BE644245, BE644246, BE644248, BE644249, BE644250, BE761007, BE761008, BE761009 and BE761010 respectively. And then, Nine novel gene fragments was found highly ekpressed during the process of U937 cells induced with IL-6 by reverse Northern Blot analysis.Base on the novel gene fragments, computer clone (contig) and RT-PCR method was used to clone the full-length cDNA and they were named STRF2, STRF6, STRF7, STRF8 and STRF11 (signal transduction related Factor). The Time Expression Pattern Analysis confirmed further that 5 novel genes related to IL-6. The STRF7 and STRF8 are expressed highly in the U937 cells induced with IL-6 detected by Northern Blot.The 5 novel genes were analyzed for PI, Mw, Amino acid content,Hydrophobicicy, protein secondary structure, motif, domain, superfamily and electronic expression pattern, Position on Chromosome by bioinformatics methods. STRF7 is partially similarity to human homeobox protein CDX-4 and Yeast transcription regulatory protein ADR6. which shows that it mayplay an transcription factor in the life of cell. The STRF8 predicted aminoacid sequence was analyzed by Bioinformatics and found that it has Thioredoxin active site and belong to Thioredoxin superfamily. These results suggested that STRF8 belong to Thioredoxin superfamily, which shows that it may play an important role in the cell proliferation.The full length of STRF8 is 3239bp including Kozak sequence and "AATAAA" tailing signal. The ORF from 3003 bp to 1915bp encodes 363 amino acids. STRF8 was expressed in E. coli and purified by TALON Metal Affinity Resin.The conclusion of our study are as follows: (1)9 novel gene fragments were found. 5 full-length genes and ORF were obtained. (2)The novel genes were proved to be ones related with IL-6. (3)Structure and function Analysis of novel genes by bioinformatics. (4)STRF8 was expressed in E. coli and purified.