Cloning Of Whole CDNA Sequence, Expression, Locating And Function Analysis Of Stress Inducible Phosphoprotein Ⅰ Gene

The human genomic draft and the scanning of genomic methylation give a clue that there is a methylation site related to leukemia on the chromosome 11. The gene of Stress Induced Phosphoprotein 1 (STIP1) is located on the downstream position. The STIP1 protein is an important molecular chaperone and it includes tetratricopiptide repeats (TPR). Some proteins in the human body also contain the similar structural motifs such as protein phosphatase 5(PP5), immunophilins(FKBP51, FKBP52, Cyp40), cyclin proteins(cdc gene), heat stress proteins(Hsp70,Hsp90). The two ends of the STIP1 protein is connected with Hsp70 and Hsp90. The STIP1 protein is thought to have a vital effect on modulating the roles of development in tumor cells, stress responses, steroid hormones and proliferation or differentiation in the cells.To clone the whole sequence of the Stress Induced Phosphoprotein 1 (STIP1), rapid amplification of cDNA ends (RACE) and long-distance polymerase chain reaction (PCR) were used to expand the target sequences of the 5'end, the 3' end and the middle in STIP1 gene. The whole cDNA sequence was tested through purification of PCR products, connecting with vectors, selection in the positive clones and sequencing. The sequence of the STIP1 gene not only included the known sequence, but also contained the new extron on the 5' end through the bioinformatics and comparing with known sequence with basic local alignment search tool (BLAST). The extron was used to design the primers which was studied in the expression of the STPlgene mRNA transcripts. The cloned whole sequence laid dependable foundation for researching the expression, location and function of the STIP1 gene in the cultured cells. The cell cycle were examined by flow cytometer and the cell numbers were reduced obviously in the stage of Go-Gi and G2-M.To investigate the expression, location and function, long distance PCR was used to expand the targets containing open reading frame. The expressive vectors of prokaryotic and eukaryotic cells were constructed after product purification and connecting with vectors. The prokayotic vectors were induced to express the protein by IPTG and the protein was seen by denaturalization PAG electrophoresis and dyeing. The eukayotic expressive vectors were transfected into culture cells and the protein was found in the nulceolus under the observation of the confocal microscope. The transfected cells were chosen by the geneticin (G418). The cell cycle was examined by flow cytometer and the cell numbers were reduced obviously in the stage of Go-Gi and G2-M, but increased distinguishably in the S stage. The STIP1 protein is a nucleus protein and a molecular chaperone which contains TPR motif. The protein may be interact with other proteins which have the same motif but have different functions. The prokarotic expressive vectors were constructed to provide the precondition in order to investigate the interaction of the STIP1 protein and other proteins. The cell numbers in the proliferative stage were increased obviously by the STIP1 protein which may be have a relation with development of tumor cells and transforming from benign tumors to malignant tumors. The function of the STIP1 protein needs studying more.To examine the expression of the STIP1 mRNA transcripts, reverse transcriptase polymerase chain reaction (RT-PCR) was adopted. However the sequences of the total 12 extrons in the STIP1 gene were matched with the DNA sequences on the X chromosome, so that primers can not be designed in the PCR reaction. The 5' end sequences were extended by bioinformatics methods and the extended sequences were an new extron of the gene. These sequences did not match with the DNA sequences on the X chromosome. The primers were designed to expand the target and the later was cloned to prove the same sequences with our assumption. And the results of rapid amplification of cDNA 5' end tested our hypothesis. The mRNA expression was different in the primary cells of leukemia.The relatively expressive quantity of the mRNA tr...