Research In The Expression And Function Of Human Proliferation-Related Kinase (Plk3/Prk)

Human Proliferation-related kinase (Prk) was cloned and characterized by Wei Dai and others in 1996. Prk is a kind of serine/threonine protein kinase and has been a new member of the Plks (Polo-like kinases) family. Members of the family mainly include Polo from Drosophila melanogaster, CdcSp in Saccharomyces cerevisiae, Plol in Schizosaccharmoyces prombe and the four mammalian genes Plkl, Snk (Plk2), Prk/Fnk (Plk3) and Sak. Prk gene localizes to chromosome band 8p21, a region that exhibits a high frequency of loss of heterozygosity in a variety of human cancers. Prk gene shares a strong overall homology to mouse Fnk and now it has been renamed as Plk3 (Polo-like kinase 3). Plk3 has been reported to be involved in regulation of cell cycle progression as the other members in the family including the regulation of microtubule dynamics, centrosomal function and the MTOC (microtubule organization center) in the cell. In most interphase cells Plk3 is mainly localized around the nuclear membrane and it appeared to be localized to mitotic apparatus during mitosis. Plk3 kinase activity peaks at the late S and G2 stages of the cell cycle and may regulate the onset of mitosis/meiosis in animal cells. Current studies revealed that plk3 mRNA expression is restricted to very limited tissues (placenta, ovaries and lung) and megakaryocytic cell lines (Dami, MO7e and glioma cell lines) at a moderate or low level. Plk3 mRNA expression is down-regulated or absent in human squamous cell carcinomas of the head and neck, lung carcinomas and the rat colon tumors induced by carcinogen. The studies of Plk3 abroad mainly focused on theexpression of it in tumor tissues, the relationship with other members of the Plks family and the mechanisms of it to regulate the cell cycle progression. We choose the aspects that have been less well defined based on the studies abroad on Plk3, so our studies were designed to investigate the possibility of expressing and purifying Plk3 protein in the prokaryotic expression system and to further evaluate the function of it in the neoplastic cells.Research objectives:1. Amplify the plk3 cDNA products and subclone it into the T-vector for DNA sequencing analysis and identifying the sequence. Construct the fusion and unfusion prokaryotic expression vectors of plk3 gene. Using temperature and drug respectively to induce the expression of Plk3 protein and compare the differences between the two ways, and then screen the suitable conditions for mass express the protein for antibodies production in future.2. Construct the eukaryotic retrovirus system of plk3 gene. Transfect the recombinant retroviral vector into packaging cells and then collect the virus supernatant to transfect the K562 and HL60 neoplastic cells. The recombinant retrovirus contained the plk3 gene is introduced into the neoplastic cells and integrated into the genome of the cells. Culture the neopalstic cells and screen the positive clones using antibiotic selection. Observe and compare the positive clones with the control of wild neoplastic cells. Analyze the influences of plk3 gene transduction on the abilities of neoplastic cells proliferation and oncogenesis in vivo.Methods and results:Amplified the full-length (about 1.85kb) plk3/prk cDNA gene in vitro using PCR and then subcloned it into pMD18-T vector for DNA sequencing analysis and restriction mapping. Target DNA fragment inpMD-18T-plk3 was digested with restriction enzymes, and then linked with expression vectors. Extracted, purified and identified the recombinant plasmids, successfully constructed the pBV220-plk3 unfusion expression vector and the pET-24a(+)-plk3 fusion expression vector. Induced the protein expression in E.coli DHSaand BL-21(DE3) by temperature and drug control respectively. There were less unfusion protein expression (10.9%) and relative higher fusion protein expression (25%) with a 6-His target in its N-terminal. Initial purification revealed that the protein mainly existed in the supernatant using the unfusion expr...