Study On Molecular Cloning, Expression And Preliminary Function Of A Novel Human Gene LACE1

Objectives: In this study, We analyze the cloning, expression and characteristics of LACE1. This study not only has important theoretical significance but also has potential value for clinical diagnosis and treatment of disease,and provide the theoretical foundations for research and development genetic drugs.Methods:The LACE1 gene order, expression spectrum and Species homology were analyzed by bioinformatics software, and the LACE1 gene in the cell locates was forecasted; the expression of the LACE1 gene was examined by RT-PCR in the different cell; Construction of molecular cloning and gene deletions of the transmembrane domain of the eukaryotic expression vector to inprove that the 293T cell lines established for the purpose of gene transfection in vitro eukaryotic expression platform; using a confocal microscope to detecte the localization of 293T cells which transfected by green fluorescent labeling with GFP fusion plasmid; The expression of the LACE1 gene on 293T cells implications was studied by conventional morphology and MTT.Results:1. By bioinformatics analysis of gene LACE1 (Homo sapiens lactation elevated 1), the GeneID: 246269; RefSeq: NM₁45315.3. The gene is 2262 bp, from 13 exons and 12 introns, location and 6q 21. from 187 1632 has a 481 amino acid coding reading frame encodes a protein of about 54 kDa. The protein isoelectric point was 7.29, no disulfide bonds.2. RT-PCR results showed LACE1 universal expression, which in HeLa cells, HepG2 cells, 293 cells with high expression, Raji cells, H520 cells, 293T cells showed low expression, suggesting that the gene may be associated with reduced cancer-related signaling pathway related.3. Biogps profile analysis and LACE1 homology with other species analysis: analysis the database by the NCBI BLASTn comparison, we found LACE1 in Homo sapien; Bos Taurus; Canis lupus fami; Mus musculis; Rattus norvegicu; Gallus gallus and many other species homology exists.4. PCR the LACE1 gene and cloned into the eukaryotic expression vector pcDNA3.1B (pcDB), proved by restriction analysis and sequencing of the gene sequences published GeneBank sequence database, indicating that pcDB-LACE1 expression plasmid was successfully constructed. Construction of the plasmid carrying the green fluorescent label pEGFP-N1-LACE1.5. LACE1 subcellular localization showed that the protein is located in the cytoplasm. 6. MTT tests showed that the gene can inhibit cell proliferation, westernblot showed that the inhibition of cell proliferation maybe related to caspase gene and this inhibitory activity was time-dependent.Conclusions:1. A novel human genes evolutionarily conserved LACE1 was found and it was located in the cytoplasm.2. RT-PCR results showed that LACE1 in a variety of normal human tissues, in most human cell lines with different degrees of expression. 293T cells showed low expression which suggest that the gene may be associated with reduced cancer-related signaling pathway related.3. LACE1 can inhibit cell proliferation in a time-dependent manner, which may relate to caspase .