The Bcl-2-associated athanogene (BAG) family proteins are BAG-domain (BD) containing protein and are evolutionally conserved among eukaryotes. BAG family proteins play a critical role in a wide variety of cellular processes, such as cell survival, proliferation, migration, apoptosis, stress response and neural differentiation. BDs are responsible in binding HSP70 to BAG proteins. However, how BDs mediate the protein complexes assembly to govern the diverse functions of BAG proteins is needed further studies. To explore the roles of BDs in BAG family proteins, GST-BDs pull-down coupled with LC-MS/MS were performed to screen the BDs associated proteins in cytosolic extracts from Hela cells. 370 potential BDs interacting proteins were identified. GO (Gene Ontology) annotation analysis shows that BDs interacting proteins function in cellular protein quality control, glycolysis, immune response, stress response, ubiquitin-proteasome regulation, cell cycle process. In particular, KEGG (Kyoto Enyoolpedia of genes and Genomes) pathway enrichment analysis reveals that BDs interacting proteins involve in several important pathways, including FGF signaling pathway, EGF receptor signaling pathway, PDGF signaling pathway and Ras pathway. In addition,different BDs can specifically binding different partners. Overall, this expands knowledge of the BDs interactome, provides a valuable resource for understanding the roles of BDs in mediating BAG proteins' different cellular functions.
It is intending to prepare tumor necrosis factor α (TNFα) derivative TRSP10 which can lead prostate cancer DU145 cells apoptosis by genetic engineering and to study the inhibitory effect on DU145 cells in vitro. Trsp 10 gene sequences were synthesized by overlap extension PCR and then inserted into the site between SapI and NdeI in the highly-efficient expression vector,pKYB-MCS and conduct the optimal conditions to induce the expression of the fusion protein and set up the technology from vector construction to the expression and purification of the recombinant bacteria. MTT detect the inhibit proliferation effect of the TRSP10 on prostate cancer DU145 cells. The results showed that the optimal expression condition was as follows: at 0.8 mmol /L IPTG,37 ℃ and 8 hours.Puried by the IMPACT system and HPLC technology, the purity of the TRSP10 is 96%.The molecular mass is 3.59kDa that is determined by the mass spectrometry, which is consistent with the theoretical data. In vitro, studies showed that, TRSP10 could significantly inhibit the proliferation of prostate cancer DU145 cells.The rates of inhibition among the concentrations of 5,10,20,40μmol/L, are 11.40%, 22.97%, 33.26%, 48.35% respectively.
Objective: Previous studies have respectively demonstrated that HIV-1 Nef down-regulated the cell surface expression of CD4, and that Nef interacted with the host protein hnRNPK. The purpose is to investigate (1) if hnRNPK regulates the surface expression of CD4, and (2) if hnRNPK is involved in the down-regulation of CD4 by HIV-1 Nef. Methods: in vitro GST-pulldown assay was used to confirm the interaction of HIV-1 Nef with hnRNPK. HIV-1 Nef was expressed in HeLa-CD4 cells by transient transfection, hnRNPK was knocked down by means of siRNA, and the cell surface expression level of CD4 was assessed by using flow cytometry. Results: (1) The GST-pulldown assay has successfully confirmed the Nef-hnRNPK interaction; (2) Nef ectopic expression resulted in about 75 percent reduction of the cell surface CD4 expression; (3) hnRNPK knockdown reduced dramatically the cell surface expression of CD4(50% of reduction) regardless Nef was present or not. Similarly, the effect of Nef on the cell surface expression of CD4 was not affected by hnRNPK knockdown. Conclusion: (1) hnRNPK interacts with Nef; (2) hnRNPK facilitates the cell surface expression of CD4, but the relationship between this effect and the down-regulation of CD4 expresion by Nef remains unclear, probably reflecting the complex regulation involving other factors.
Objective: To explore whether transient receptor potential canonical 3 (TRPC3) plays a role in the apoptosis of oligodendrocytes induced by oxygen glucose deprivation(OGD).Methods: Control group were established by primary cultured oligodendrocytes of SD rats,model group were established by primary cultured oligodendrocytes of SD rats which exposed to OGD2h,treated group were established by model group+ Pyr3.Western blotting were used to detect the expression of TRPC3 at protein levels.The viability of the oligodendrocytes was determined by MTT assay,and the apoptosis of oligodendrocytes were measured by flow cytometry after AnnexinV-FITC staining,intracellular Ca2+ concentration were measured by flow cytometry and Laser Confocal Scamfing Microscope(LCSM).Results: Specific markers of A2B5 and MBP in oligodendrocytes were positive and the purification is more than 95%,OGD model was established successfully.A significant increase in the expression of TRPC3 in cultured oligodendrocytes.The viability of OGD oligodendrocytes (54.34±6.55)% was obviously decreased and the apoptosis rate was increased (24.24±0.86)% when compared with the control cells (P<0.05).Pyr3 significantly increased the survival rate (72.26±5.41)% and decreased the apoptosis rate (14.82±0.28)% and partially suppressed Ca2+ in oligodendrocytes (P<0.05).Conclusion: The increased expression of TRPC3 which mediated intracellular calcium level may be one of the important reasons for OGD oligodendrocyte apoptosis.
HPD is a kind of tyrosine metabolizing enzyme with specific expression in liver. The current research mainly focuses on the relationship between HPD and tyrosinemia diseases, while there is less reporter about the transcription regulational mechanism of HPD, and the mechanism of HPDliver specific expression is not yet known. The aim was to construct HPD gene promoter luciferase reporter gene vector, analyze its transcriptional activity in the human hepatocellular cells, and begin to clarify the regulational mechanisms of its specific expression in liver. Methods: The 5′flanking sequence from －2 000 to +39 of the human HPD gene was analyzed by the UCSC software for the promoter character. This sequence were cloned from the human hepatocellular carcinoma cell HepG2 genomic DNA by PCR and inserted into the pGL3Basic vector.Through the design of different primers, a series of 5′deleted constructs were made to product seven luciferase reporter gene vectors in different lengths. Then the seven reconstructors were transfected into HepG2,L02 and HEK293 cells respectively to determine the transcriptional activity of HPD gene promoter by the dualluciferase analysis.Results: The dualluciferase analysis results showed that the fragment from －600 to －400 had a stronger transcriptional activity in HepG2and L02 than in HEK293,while the fragment from －400 to +39 had the transcriptional activity in all of the three cells. Bioinformatics analysis results showed that there are many liver specific transcription factor binding sites in －600 ~－400 area of HPD gene promoter sequence. Conclusion: The luciferase reporter gene vectors with the HPD gene promoters are constructed successfully. In the fragment from －600 to －400,there would have the key regulatory elements for the HPD liverspecific expression,which provides an important basis and lays a theoretical foundation for further revealing the transcriptional regulational mechanism of HPDliver specific expression.
Objective: The fusion protein HBV pre-C protein - mouse IgG Fc (HBV pre-c-Fc) protein was expressed based on the baculovirus expression vector system, and identify its immunogenicity. Methods: target gene HBV pre-c-Fc connected to pFastBac1 vector, pFastBac1-HBV pre-c-Fc plasmid was transformed DH10Bac competent, transposase Bacmid through Th7 transposon into the target gene, got Bacmid-HBV pre -c-Fc shuttle vector, Cellfectin ⅡReagent transfected Sf9 insect cells to obtain P1 generation virus, transfected Sf9 repeated to obtain high titer virus. Cell supernatants were collected after ultrafiltration to obtain the target protein HBV pre-c-Fc purified by Protein G affinity chromatography. The purified protein thigh intramuscularly immunized BALB/c mice and detected the concentration of serum hepatitis B virus core protein antibody. Results: HBV pre-c-Fc successfully expressed in insect cells, the purity of purified protein was over 90%, the production of protein is about 3.03mg/L, purified protein can effectively immunize the BALB/c mice to produce specific antibodies. Conclusion: The expression of immunogenic HBV pre-c-Fc protein based on the baculovirus expression vector system can be the production of hepatitis B therapeutic vaccine foundation.
The aim is to express and identify the fusion protein cTnI-linker-TnC, and detect its immunogenicity for preparation of monoclonal antibody of anti-cTnI-TnC. A short DNA sequence which codes 15 neutral amino acid residues was used to link cTnI and cTnC. Then, the recombinant prokaryotic expression vectors pET32a-cTnI-linker-TnC and pET28a-cTnI-linker-TnC were constructed to express the fusion protein cTnI-linker-TnC by transformed into E.coli BL21 cells respectively. And the fusion proteins cTnI-linker-TnC were identified by 12% SDS-PAGE and indirect ELISA test.At the same time, the concentrations of the inducing agent,expression times and temperatures were optimized to obtain high soluble target protein. At last, the commercial monoclonal antibodies of anti-cTnI and anti-cTnC were used to do the Western blot test and double antibody sandwich ELISA,and the serum of myocardial infarction patients were used to do the indirect ELISA. The results showed that the fusion protein cTnI-linker-TnC has a high soluble expression, and has reserved its immunogenicity well. This indicates the fusion protein cTnI-linker-TnC may replace the natural cTnI-TnC compound in the future.
To further investigate the function of non symbiotic hemoglobin of spinach (SoHb), the full length cDNA encoding SoHb was amplified by RT-PCR from spinach root and cloned into inducible expression vector pET32a. The recombinant prokaryotic expression plasmid was transformed into E.coli BL21 star (DE3) strain for genetic engineering strain. The transformed strain was induced with isopropyl-beta- -D-thiogalactoside (IPTG) for expressing fusion protein. SDS-PAGE analysis showed that the recombinant protein with a molecular mass about 38 kDa was highly expressed in E.coli and presented both in the supernatant and the pellet part of E.coli lysates. The pET32a-SoHb strain was more resistant to nitrosative stress than the pET32a empty vector strain. The supernatant was further purified by Ni2+ NTA affinity chromatography and immunized white mouses as antigen. The polyclonal antibody was obtained and analyzed by Western blot. It laid foundation for investigating the function of SoHb.
Keto acid decarboxylase is the key enzyme for biological synthesis of isoamyl alcohol, rarely existed in E. coli. Keto acid decarboxylase gene kivD(rbs) obtained from Lactococcuslactis subsp.Lactis genome DNA through reaction of PCR, was inserted into E. coli high expression vector pET-28a(+). The formed pET-kivD(rbs), was then transformed into E. coli BL21(DE3) by hot blow. The activity of keto acid decarboxylase and isoamyl alcohol were detected in the fermentation broth.
Objective: To make the efficient detection of malignant tumor metastasis rate in cancer metastasis in animal models by use of the multiplex real-time fluorescence quantitative PCR. Methods: The species-specific primers and probes for detection of beta-2-macroglobulin(β2m) gene of human beings and mouse in multiplex real-time fluorescence quantitative PCR assay was designed. Total DNA as templates were extracted respectively from human ACC cells and mouse Sp2/0 cells. The specificity and sensitivity of these primer pairs were separately confirmed using simplex real-time PCR analysis. The specificity, sensitivity and accuracy of the multiplex real-time fluorescence quantitative PCR method were evaluated and compared with the traditional method by weighing tissues of tumor metastasis to calculate the rate of tumor metastasis. Results: The limit of detection (LOD) of the assay was 0.006ng/μl DNA for each target species (1:104 dilution). Conclusions: This system proved its accuracy, precision and applicability for the detection of malignant tumor metastasis rate in animal models.
To generate F-box and Leucine-rich repeat protein 15 conditional knockout mice model,and confirm the genotype for further functional research. Using KO first strategy,to construct the conditional knockout vector targeting exon2 and exon3.In additional, FRT-SA-IRES-lacZ-loxp-neo-loxp element between intron 3 and intron 4 was also inserted.By Long Range PCR and Southern blot methods the positive clones were selected. Therefore the dual selected ES cells were microinjected into blastula of C57BL/6J mice, then blastula transplantations into the host mice,Chimerical mice were generated and the FBXL15-Lox Ptargeted mice were identified by PCR method. This animal model established the foundation for further study of FBXL15's pivotal roles in regulation upon embryos development and bone metabolism
New erythropoiesis stimulating protein (NESP), is a highly glycosylated rhEPO analogues containing five N-terminal sugar chain and twice as high in sialic acid residues than that of rhEPO. NESP has greater metabolic stability and three times the half-life of rhEPO. Fusion with Fc fragment can prolongs half-life of a protein in vivo. NESP was used to construct the new erythropoiesis stimulating protein-IgG2 Fc fragment fusion protein (NESP-Fc).After process optimization, the expression of NESP-Fc is about 1.4g/L. A series of studies confirmed that the fusion protein can form dimer, and the molecular weight of the dimer is about 130kDa. The NESP-Fc fusion protein can significantly promote the growth of UT-7 cells in vitro, and fusion with Fc didn't impact the biological function of NESP. In mouse model, NESP-Fc dose-dependently increased reticulocyte level. The half-life of NESP-Fc fusion protein can reach up to 56 hours in rats. These studies of NESP-Fc fusion protein proved that NESP-Fc could be potentially used as a medicine for anemia, and it also laid a solid foundation for future clinical trial and the final industrialization.
Aptamers is a kind of oligonucleotides which are isolated through a process termed systematic evolution of ligands by exponential enrichment. Because of its peculiar molecular recognition ability and the specific structure, aptamer is one of the most prospectly biological molecules. An overview of recent developments in aptamer nanomaterial hybrid was presented.
Streptomyces, a kind of Gram-positive bacteria, can produce lots of secondary metabolites which are widely used in the pharmaceutical industry, food processing and agriculture production. Genetic manipulation for Streptomyces is the foundation for the discovery and development of new secondary metabolites. The emergence of synthetic biology opens a new window for researches in Streptomyces and achievements on cloning and assembly of the biosynthetic gene cluster, the chassis cell design and the fitness were reviewed.
Endophytic actinomycetes are special groups of microorganisms,associated with the host plant with symbiotic relationship, which have amazing variety of biological function, and can produce lots of potentially biological active substances. The relationship between the Endophytic actinomycetes and the host plant was described, and then the progress in their biological effects on improvement of plant stress resistance, promotion of growth and biological nitrogen fixation were introduced. The antibiotics, enzymes and anti-tumor activity of bioactive substances produced by Endophytic actinomycetes, and the future prospects of these substances in agriculture, medicine and food industry were summarized. Finally, the problems and trends about the biological effects of Endophytic actinomycetes and their bioactive substances were also analyzed.
National major special project for the development of transgenic organisms was set up in 2008. At the same time, the funding agency supported a research project named "Study on reference materials for genetically modified organisms (GMOs) detection". Besides the concept and function of reference materials for GMOs, its varieties and its advantages and disadvantages were also introduced. At last, elucidated research actuality and expectation of focus on the development of reference materials for GMOs in the future.
