Clone,Analysis And Function Of A Novel KLP Gene In Zebrafish

ObjectiveScreening or identifying and clarifying some novel genes associating to tumors , virus infection and embryo development based on the rapid development in the genomics and bioinformatics fields.MethodsScreen, Clone, analyze and study novel genes by performing cross - immunomic analysis of SEREX (Serological analysis of recombinant cDNA expression library) and database mining. cDNA expression library derived from zebrafish heart oocytes was cross-immunomic analyzed by an antibody pool against all antigenic proteins of HUVECs . Insert sequences in posinive phage clones were then transformed to phagemid and sequenced and analyed to decided to further study or not. Full-length genes and complete ORFs were obtained by using insilicom strategy , including est assembling direct prediction from genome sequences .The predicted results were validated by RT-PCR and sequencing .Results1. Cross-immunomic screening:We prepared an antigen pool from differentiated poorly zebrafish oocyte,and prepare an antibody pool against all antigenic proteins of differentiated well HUVECs. Finally, we got 22 positive clone.2. Full-length/ORF clone and bioinformatics:We firstly clone in silicon the cmplete ORF of KLP based on genomic sequence and then validated it with RT-PCR and sequenceing. 3. Functional studies:We got a gene of 5171bp clone 1574 amino acid residures containing complete ORF by prediction directly from corresponding genomic sequence and assembling. The protein contains 8 C2H2 motifs and has high similarity with KAISO and KAISO, however, without BTB/POZ conserved domain. This indicated that it is probably a novel transcripter and so named KLP.ConclusionCross-immunomic screeing was effective and economical to identify some novel or old antigens that could elicit cross-immune reaction;the combination of direct prediction, EST assembling and RT - PCR verification was effective to in silicon cloning complete ORF or full-length gene for species whose genome sequence were known. Detailed bioinformatics analysis is powerful to reveal functions of previously functional unknown genes.