Screening And Analysising New Binding Protein (Nbp), A Novel Interactive Protein With P12^CDK2AP1 , By Yeast Two-hybird Technique And Bioinformatics

Cancer is a serious threat to human health and even the deadly malignant disease, its incidence increased year by year as well as a young trend, and cancer has always been one of the important topics in medicine. Oral cancer ranking sixth in the world cancer incidence, and because of it's special area, oral cancer seriously impact the of patients' life and survive. In recent years, tumors mechanism and research with cancer gene, tumor-suppressor genes interacting relationship at the molecular level has become one of the main direction in tumor pathogenesis and gene therapy research . Current research results have indicated that the occurrence of cancer and cancer gene expression and tumor-suppressor genes lost expression are closely related. CDK2AP1 (cyclin associating protein kinase 2 dependent gene is a 1) is an oral cancer incidence relevant candidate tumor-suppressor gene. CDK2AP1 gene originally called DOC - 1 (deleted in oral cancer-1), which was found, separated and the confirmed as a candidate for tumor-suppressor genes by Todd in 1995 in oral cancer model in hamsters. P12CDK2AP1 protein is its coding protein, has the role of suppress tumor growth. CDK2AP1 gene is located in 12q24 human chromosomes, coding 115 amino acids, its expression protein p12^CDK2AP1 the molecular weight of 12.4 kDa. In the research of CDK2AP1 in hamster model, CDK2AP1 can induce cell apoptosis of the oral malignant keratinocyte .Through down-regulating CDK2 (cut protein cell cycle dependence protein kinase 2) activity and the expression level, its expression p12^CDK2AP1 weaken the Rb gene suppression, plays the role of inhibiting tumor cell growth. In squamous cell cancer cells ectopic expression, after the P12CDK2AP1 protein with CDK2 and DNA - polymerase alpha (DNA polymerases alpha) union, it inhibit the these two kinds of enzyme activity and change the malignant cell phenotype . At present, mechanism study of significantly reduced even lack of p12^CDK2AP1 protein expression in oral squamous cell carcinoma the was not reported, and its interaction proteins and its protein network still not very clear.Our study adopts yeast two-hybrid technology to screen protein interact with p12^CDK2AP1 in normal liver double cDNA library, thus to find p12^CDK2AP1 protein interactions with the related factors, then we adopts bioinformatics analysis and prediction by means of interaction proteins, to further illustrate different types of CDK2AP1 genetic mechanism of the effects of CDK2AP1, research p12^CDK2AP1 protein function, reveal the role of foundation network.Objective:The purpose of this research is through yeast hybrid technology in normal liver double cDNA library screening and p12^CDK2AP1 protein in the interaction of protein, thus further research p12^CDK2AP1 protein in the related factors, and lack of oral role network. Use bioinformatics method analysis of the different types of interaction proteins. To further illustrate CDK2AP1 genetic mechanism of the effects lay the foundation.Method:1, optimize existing yeast two-hybrid screening work system, make its further effective and accurate, provide favorable conditions for the subsequent screening.2, use molecular cloning p12^CDK2AP1 protein in the reorganization of yeast two-hybrid bait plasmid, and verify its practicability. Meanwhile lay the foundation by amplify normal liver cDNA library for further p12^CDK2AP1 protein interaction proteins screening .3, use yeast two-hybrid technology to screen protein interact with p12^CDK2AP1 protein in normal liver cDNA library.4, using the method of molecular clone to restructure the original nuclear expression plasmid of the interaction proteins and induce its expression. Restructure p12^CDK2AP1 protein eukaryotic expression plasmid expressing, and induce its expression.5, use bioinformatics technology related database and software to forecast and analyze the information of interaction proteins. Including interactions protein species classification, genome positioning, physical and chemical properties of interactions, transmembrane characteristics, sub-cellular localization, signal peptide expression, secondary structure, the possible functions of the base sequence and function of homologous protein and the possible sites between than the homology.Results:Using the yeast two-hybrid method, we found a new protein (GENE ID LOC93622,hypothetical protein BC006130), interact with p12^CDK2AP1 protein in normal liver cDNA libraries, the protein with length of 119 amino acids, temporarily named Nbp. Through the yeast two-hybrid rotary experiment and NCBI blast, excluding experimental results of false positive, yeast protein interference, wipe off none-coding areas and confirmed its cDNA sequence a human DNA pac with 100% coincidence rate in DNA sequence, the chromosomal locates in human chromosome 4. We find the chromosomal location, nucleic acid sequence, MORF4 protein (amino acid sequence mortality factor 4) similar, may be related to MORF MRG (those with a certain't) protein family . Using the method of bioinformatics we analyzed and predicted the physical and chemical properties, secondary structure, sub-cellular localization, signal peptide secretion and its existing in the amino acid sequence of the possible functions of the site of Nbp protein. And we successfully reorganized the Nbp protein eukaryotic expression plasmid.Conclusion:Using the yeast two-hybrid method, we successfully found a new protein Nbp interact with p12^CDK2AP1, through bioinformatic, we predicted the physical and chemical properties, secondary structure, sub-cellular localization, signal peptide secretion and its existing in the amino acid sequence of the possible functions of the site of Nbp protein. Using molecular cloning, we successfully reorganized Nbp protein eukaryotic expression plasmids, laid a foundation for further research on CDK2AP1 genes, p12^CDK2AP1 protein and their interactions, protein function and role network .