The Bioinformatic And Preliminary Functional Analysis Of A Novel Gene AK078438

Objectives: The gene AK078438 is one of the unknown function gene,which associated with vascular endothelial cell and was obtained from using TBLASTN program(protein query vs. translated database). To investigate further function of the gene and find the objective of antineoplastic angiogenesis therapy, bioinformatic analysis as well as fundermental functional analysis should be performed.Methods: Reverse transcriptase-polymerase chain reaction(RT-PCR) technique was applied to amplify the open reading frame of AK078438 gene,which could be used for identification the Web's sequence. The differation and conservation of evolutionary process could be found by further study of phylogenetic tree of the putative protein families and alignment of the amino acid sequence comparison.We clarified the gene situate in somewhere of mouse genome by using GENSCAN and TWINSCAN softwares. Domain analysis by RPSBLAST program,signal peptide by SignalP 3.0 Server, transmembrane by TMHMM-2.0 Server and hydrophobicity shows the protein physical and chemical function. Chemical modification sites of the protein on SMART Server analysis show that it would participate in enzyme regulation. At mRNA expressing levels semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)was employed for the investigation of its distribution in normal rat tissues, tumor cell and mesenchymal stem cell(MSC)lines. The eukaryotic expression green fluorescence protein(GFP)fusion protein vector was transfected into cells and identified subcellular localization. Prokaryotic expressing vector,identification of molecular weight and affinity purification were used to get the fusion protein.It would be the basis of further molecular pathway investigation and polyantibody preparation.MTT and flow cytometry were used for identification apoptosis function of the gene.Results: The full open reading frame of the gene with 1065bp,355aa were identified. Molecular weight of prokaryotic expressing protein is conform with theoretical calculation.The gene has higher homology and genera relationship on evolutionary process. It has eight extrons and seven introns and situates in 4qA1 chromosome of mouse genome.Further study show that the protein have some chemical modification,BAR,DUF1208 conserved domains and not sigal pepetide mRNA of the gene distribute in brain,uterus,colon, bone marrow,testis,kidney and without in stomach,liver,lung,spleen, heart. Rat tumor cell lines C26,B16,LL/2 and MSC have the gene and without in NS-1,Hepa. The protein is localized in cytoplasm.Some apoptosis function were got by MTT and flow cytometry, but further investigation of the function must be identificated with the result.Conculsion: To explore its possible function and information by bioinformatics methods,Protein expressing,expressing profile, the study of apoptosis function and subcellular localization of AK078438 gene set the basis for further exploring its functions.