Prokaryotic Expression And Functional Characterization Of α-1B Glycoprotein Precursor Gene And The Target Expression Of CYP4F2 Gene In Kidney

IntroductionWith the completion of Human Genome Project, the exploration of the function of human genes is undoubtedly a rough task in genetic research field. Alpha -1B -glycoprotein was isolated from normal adult plasma by Ishioka in 1986. Humana - IB glycoprotein precursor gene (α - 1BPG) was cloned from the fe-tum liver cDNA library in 2001 by our group, which provide the sequence basis for its functional research. In our previous study, the analysis of expressional profile and regulation of α - 1BPG, suggested that α - 1B glycoprotein may probably participate in the regeneration process of the liver cell and promote the proliferation of the cells. As it is very difficult to purify alpha - 1B - glycoprotein from the plasma or obtain effective expression of alpha - 1B - glycoprotein using the Pichia pastoris expression system, alpha - 1B - glycoprotein was expressed in E coli. through DNA recombination. To detect the effect of alpha -1B- glycoprotein on cell proliferation, Bel7402 cell was stimulated with rena-tured alpha - 1B - glycoprotein, and then proliferation of the cells was analyzed by Flow Cytometry and MTT. Meanwhile, recombinant alpha -1B- glycoprotein will be greatly helpful in preparing its antibody, obtaining of its downstream protein and learning the alterations of different proteins.Materials and methodsα -1B glycoprotein precursor gene was amplified by PCR. The recombinedexpression vector pET28 - a ( + ) - α1 - B was created, then transformed com-pletent BL21 ( DE3). After α - 1B glycoprotein was induced by IPTG,inclusion protein was extracted,purified and renatured. Western Blotting was used to test the purified protein. The cell proliferation was analyzed by Flow Cytometry and MTLResultsThe expression vector pET28 - a ( + ) - α1 - B was identified by restriction enzymes digestion and sequencing. The results of electrophoresis showed that the fragment produced was about 1603bp, consistent with expectation. Western Blotting shows that a protein with molecular weight about 58 kD was observed on the PVDF membrane. The protein was coincident with the expectation. After Bel7402 cell was stimulated with recombinant alpha -1B glycoprotein, cell proliferation was not found by Flow Cytometry and MTT.Conclusion1. successfully expressed and purified alpha - 1B glycoprotein in E coli.2. alpha -1B glycoprotein have no the function of promoting the cell proliferation.IntroductionWith the accomplishment of the map of the Human Genome, scientific community has entered the post - genomic epoch - Functional Genomics. Transgenic technology and gene - knockout technology has become an indispensable experimental approach for gene functional analysis in the post - genomic epoch. The most effective methods to identify gene function is to examine the phenotypic alteration of cells or the whole body after gene expression is increased or blocked.CYP4F2 is an potent AA ω - hydroxylase which can hydroxylate AA to 20 - HETE . 20 - HETE is a natriuretic and vasoactive eicosanoid and elicits specific effects on kidney vascular and tubular function that, in turn, influences blood pressure control. We analyzed that up - regulating the expression of CYP4F2 gene would result in the elevation of blood pressure, but its merchanism still remains unclear. In this research plasmid pK - CYP4F2 containing 224bp KAP promoter fragment was constructed to investigate whether KAP promoter can specificly promote the expression of CYP4F2 gene.Materials and methodsAfter the KAP promoter fragment consisting of 224bp was amplified by PCR, the luciferase report gene vector pGL3 -KAP224 was constructed. C0S7 Cells were transiently transfected with pGL3 - KAP224 and pRL - TK. Firefly and Renilla luciferase activities were measured by using the Dual - Luciferase Reporter Assay system.CYPAF1 gene was obtained by RT - PCR and ligated with the KAP promoter fragment to generate the kidney target expression vector pK - CYP4F2. After...