Full-length CDNA Cloning Of A Novel FXYD6 Gene Related To Cholangiocarcinoma With Subcloning, Bioinformatic Localization And Prokaryotic Expression Of Its Functional Domain

CCR-L2/fxyd6 gene is a novel cholangiocarcinoma related and fxyd6 homologous gene- containing L2 cDNA, which was first named and studied by us in 2002. The reference sequence of fxyd6 gene was derived from multiple replicate ESTs and validated by human genomic sequence. This gene encodes a member of a family of small membrane proteins that share a 35-amino acid signature sequence domain, beginning with the sequence FXYD. The approved human gene nomenclature for the family is FXYD-domain containing ion transport regulator. But this gene product, FXYD6, is novel and has not been characterized as a protein. Also its bioactivity and mechanism remain unclear.Study of genes related to cholangiocarcinoma is one of the most important part of the basic research in biliary diseases. Many evidences have shown the importance of ion channel protein in regulation of cholangiocyte physiology. So it is interesting and necessary to get more knowledge of the FXYD-domain containing ion transport regulator 6.To explore the characteristic and protein expression of fxyd6 gene, a new strategy is used in this study with the integration of bioinformatics and molecular biology. Bioinformatical methods were used to analyze the full-length sequence, and predict the functional domains of its protein. And the full-length sequence of fxyd6 was isolated by polymerase chain reaction from fetal hepatic and brain cDNA library, then were cloned in pGEM-T vector for sequence analyzing. The sequence of functional domain without signal peptide was designed and obtained by polymerase chain reaction, then the recombinant expression plasmid pBV220/fxyd6-ex was constructed to express human fxyd6 functional domain in the host bacteria E.coli. A series of experiments on fxyd6 were stated in the following three parts.A. The full-length sequence of fxyd6 was isolated by Touch-down PCR from fetal hepatic and brain cDNA library, which built a good basement for the followed studies. But it is unclear for the negative result, which should be got of the segment of the gene in spleen cDNA library as the GenBank displayed.B. The ORF Finder software was used in NCBI website to find the most probable encoding regions of fxyd6 gene. And we selected the +1 phase as the template sequence, from 67bp to 354bp, to predict the functional domains. The signal peptide was located from 1aa to 17aa, and the main domain was composed from 18aa to 34aa. The region between 35aa and 57aa is the transmembrane region. The FHYD peptide chain is the highly conserved amino acids.GOLDKEY Sequence Analyzing Software was used to analyze the sequence of candidate domain without signal peptide, and a pairs of primers (P5/P6) were designed with two restriction sites of Pst I and EcoR I . Code ATG was added before the beginning base of domain sequence. The objective sequence(fxyd6-ex) was obtained by polymerase chain reaction, and sub-cloned in pGEM-T vector.C. The recombinant expression plasmid pBV220/fxyd6-ex was constructed to express human fxyd6 functional domain in the host bacteria E. coli DH5 α ,...