Cloning Of Promoter Of Human Qki And Its Transcriptional Regulation By TNF

The quaking gene (qki) encodes a highly conserved RNA binding protein (QKI), which is a member of the"STAR"(signal transduction and activation of RNA) family of RNA binding proteins. quaking has a critical role in Embryonic and cardiovascular development besides myelination.some homozygous mutant quaking mice died prior to birth.becanse of the defect of cardiovascular and muscle development and others have severe central nervous system dysmyelination causing quaking movements during normal motor activities from10 days postnatal and tonic convulsion when growing up.The length of quaking gene is about 65kb,there are at least 5 diferrent isoforms of QKI producded by alternative splicing of qki transcripts in which 9 exons are included. qki-5 (5kb),qki-6 (6kb) and qki-7 (7kb) are three major transcripts of 5 and expressed as QKI-5 ,QKI-6 and QKI-7 respectively. These isoforms (QKI-5,QKI-6, and QKI-7) are identical in RNA binding motifs called KH domains as well as SH2 and SH3 domains but with the exception of the carboxy-terminal ends, which result in nuclear localization of QKI-5 and cytoplasmic localization of QKI-6 and QKI-7.Pilotte found that only one QKI isoform, QKI-7, can induce apoptosis in fibroblasts and primary rat oligodendrocytes and the unique C-terminal 14 amino acids of QKI-7 confers the ability to induce apoptosis. The coexpression of either QKI-5 or QKI-6 with QKI-7 caused the nuclear translocation of QKI-7 and suppressed the apoptosis normally observed with the expression of QKI-7. Many other researches about the fuction of QKI focus on nervous system especially on the molecular mechanism associated with medullary sheath development.NFKappaB as an oxidation-reduction sensitive transcription factor is very necessary in the development of embryo .It also can regulate the expresions of many genes associated with inflammation. Activation of NFkB induces gene transcription that promotes inflammation, such as leukocyte adhesion molecules, cytokines, and chemokines.So NFKappaB is important for initiation and progression of pathogenesis in atherosclerosis, inflammatory bowel disease, autoimmune arthritis, glomerulonephritis,asthma, lung fibrosis, septic shock, carcinogenesis and AIDS. On the other hand,mutation in some regions of qki gene can cause severe developmental defection of cardiovascular system in embryo. Next, through the analysis of qki promoter several NFKappaB binding sites can be found.Lastly qki as a RNA binding protein was predicted to recognize many putative inflammation associated targets such as: VCAM and COX-2.But the molecular mechanism about the role of quaking in inflammation and initiation, development of cardiovascular system is not clear at all.In order to detect the change of qki expression level under different stimulus in the initiation and development of inflammation and cardiovascular system.we firstly constructed the luciferases report system of qki promoter and three truncations. Then,by use of RT-PCR assays, we further observed the change of qki transcriptional level under the stimulus of TNF. At last, we successfully expressed and purified QKI-7 and developed the Polyclonal antiQKI antibody with high titer and specificity. the Polyclonal antiQKI antibody targeting QKI-7 can recognize different isoforms of QKI ,because different isoforms of QKI are identical in 90% amino acid sequence. we also use the Polyclonal antiQKI antibody to detect the change of expressional level under TNF stimulus.In addition,we compared the effects of three different immunoadjuvant in producding Polyclonal antiQKI antibody.The main results are as follows:1.Cloning of the promoter of qki and constructing of promoter luciferase report vector.We made the luciferase report system by cloning the upstream sequence of qki gene (2165bp from ATG), into the multicone site of pGL3-Basic vector containing the downstream luciferase gene, then, we could detect the luciferase activity if the promoter was functional.We transfected luciferase report system into the Hy906 and 293 cell line, then detected the rise of luciferase reading. The results demonstrated that the functional promoter of the qki has been cloned in the report vector. The activity of qki promoter was downregulated under the stimulation of TNF and P65 subunit of NFKappaB and the down regulation of qki promoter activity by TNF was inhibited when vector expressing IkappaB mutant form was co-transfected.2.construction of three different promoter truncations and analysis of promoter activity by stimulation of TNF and inhibition of TNF associated signal moleculars.We cloned three truncations of qki promoter 5'end (QP1 968bp, QP2 1083,QP3 1652bp including 1,2 and 3 NFKappaB binding site respectively) according to the predicted NFKappaB binding sites in 2165bp from ATG..Then we transfected these luciferase report systems into cell line, the luciferase activities of three delection vectors were decreased after use of TNF and active subunit P65. The promoter activities of QP4 and QP1 can be inhibited by IkappaB mutant form,so we proposed the upstream region (-968bp from ATG) is the shortest sequence in which active NFKappaB binding site is included.3.RT-PCR result showed the transcriptional level of qki decreased at the early time then increased under the stimulation of TNF in 24 hours.4.Western-blot result showed the expressional level of qki decreased gradually under the stimulation of TNF in 16 hours. 5.prokaryotic expression of QKI and production of Polyclonal antiQKI antibody. PET32b(+) vector containing QKI-7 coding sequence was transfected into competent BL21(DE3) bacteria by heat shock 6×His -QKI-7 fusion protein induced by IPTG in E. Coli was purified with anti-his affinity chromatography, hydrophobic chromatography and negative ion exchange chromatography. After verified by SDS-PAGE,Western blot, the purified 6×His -QKI-7 fusion protein respectively mixed with Freund'adjuvant , polyacrylamide and NC membrane was used to immunize rabbit . The rabbit anti QKI serum was harvested. The Polyclonal antiQKI antibody targeting QKI-7 can recognize different isoforms of QKI ,because different isoforms of QKI are identical in 90% amino acid sequence.The titer and the specificity of the ployclonal anti QKI antibody were determined by Western blot.In summary, we obtained the region of qki promoter and made the luciferase report plasmids. Then by use of luciferase reporter assay,RT-PCR and Western blot we detected the transcriptional regulation of qki by TNF.In order to study the change of expressional level of qki under different stimulations in the initiation and development of inflammation we expressed and purified 6×His -QKI-7 fusion protein then developed Polyclonal antiQKI antibody with high specificity.