Objective: To study the molecular mechanism of TSA-induced indoleamine 2, 3-dioxygenase (IDO) downregulation in human liver cancer cell line HepG2. Methods: The roles of TSA on the IFN-γ induced IDO expression in HepG2 cell, the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and the activation of interferon regulatory factor 1 (IRF-1) were examined by western blotting. IDO expression in TSA-treated HepG2 cells was also detected by immunocytochemistry, and changes in the expression of IFN-γ receptor 2 was analyzed by Flow cytometry. Effect of TSA on STAT1 nuclear translocation was observed by a confocal laser-scanning microscope. The Luciferase activity of the activation of γ-interferon activated sites (GAS), interferon stimulated response element (ISRE) and nuclear factor-κB (NF-κB) was measured by Dual Luciferase Reporter Assay System. Results: TSA dose-dependently reduced IFN-γ induced IDO expression, and inhibited STAT1 phosphorylation at Tyr-701 and nuclear translocation in HepG2 cell,but it upregulated the expression of IFN-γ receptor 2. Dual luciferase reporter assay and Western blotting results showed that TSA blocked IFN-γ-induced activation of GAS and IRF-1, but not NF-κB and ISRE. Conclusion: TSA can down-regulated IFN-γ induced IDO expression in HepG2 cell, which may be associated with the repression of phosphorylation and nuclear translocation of STAT1 and its binding to GAS, not due to reduced expression of the IFN-γ receptor.
The production of recombinant antibody was significantly enhanced by the method of overexpression of Heat Shock Protein 70 in Chinese Hamster Ovary (CHO) cells. The gene HSP70 was cloned from CHO/dhfr-cells, then HSP70 expression plasmid pcDNA3.1-HSP70 was constructed and transfected into CHO/dhfr-cells. CHO cell line overexpressing HSP70 stably was isolated and the expression of HSP70 gene was analyzed through RT-PCR. Anti-HBs chimeric antibody expression plasmids were transfected into the HSP70 cell line, ELISA was performed to test the production of anti-HBs antibody. It demonstrated that the overexpression of HSP70 can enhance the production of recombinant antibody in CHO/dhfr-cells (P<0.05).
Objective: To construct and verify the neotype gene vaccine eukaryotic expression vector pVAX2 which contains two gene expression cassettes. Methods: The new expression cassette was designed and obtained by artificial chemical synthesis that contains eight elements—Human cytomegalovirus (CMV) promoter, T7 promoter, signal peptide sequences, Hemagglutinin A epitope genes (HA), multiple cloning sites region(MCS),c-myc epitope, Platelet-derived growth factor receptor transmembrane domain (PDGFR-TM) and Bovine growth hormone polyadenylation (BGH polyA). And the restriction enzyme NruⅠwas added to its upstream and downstream, and then it linked with cloning vector pGH to construct pGH-Ⅱ. The pGH-Ⅱ was cutted with NruⅠ,and the fragment (1 300bp) was reclaimed and cloned into the eukaryotic expression vector pVAX1 after removing phosphoryl groups with Alkaline phosphatase to construct the neotype gene vaccine eukaryotic expression vector pVAX2, which was identified with NruⅠand BglⅡ/PstⅠ. To identify the expression ability of pVAX2, the enhanced green fluorescent protein (EGFP) genes as report gene was inserted into the difference cassette of the two expression cassettes, and then transfected into BHK-21cells by liposome. The function of pVAX2 was verified by the reverse-transcriptase-polymerase chain reaction (RT-PCR) and fluorescence microscope. Result: The expression of EGFP in both expression cassettes was high efficiency in BHK-21 cells, and didn’t impact each other. Especially, the new expression cassette allowed display of proteins on the cell surface. Conclusion: The eukaryotic expression vector pVAX2 is successfully constructed, which could be used for the research of polyvalency DNA vaccines.
Objective: The aim is to construct lentiviral vector containing GFP reporter gene driven by human telomerase reverse transcriptase(hTERT)gene and to establish high-titer lentiviral packaging system, then observe the expression of hTERT gene. Methods: Digested the plasmid PCI-neo-hTERT with double enzyme digestion and subcloned hTERT into the lentiviral vector, pCDH-CMV-MCS-EF1-copGFP,to generate the lentiviral expression vector, pCDH-hTERT. The accuracy of hTERT fragment was confirmed by double enzyme digestion and DNA sequencing analysis. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pCDH-hTERT,with the packaging plasmids pCDH-PACK-GAG、pCDH-PACK-REV and VSV-G which are the 3rd generation of lentiviral vector systems containing green fluorescent protein (GFP)gene. Virus supernatant was collected and concentrated, then the titer of virus was tested. The resulting recombinant lentiviruses which carrying hTERT were then used to infect target cell lines. GFP, hTERT mRNA and telomerase expression in 293T and hUVEC were detected by fluorescent microscope, RT-PCR,Western blotting and TRAP-PCR-ELISA. Result: Plasmid pCDH-hTERT carried the correct hTERT gene. The recombinant lentiviruses pCDH-hTERT which carried hTERT could be produced by co-transfection of pCDH-hTERT and packaging plasmids to 293T; The recombinant lentiviruses which carried hTERT could infect and deliver hTERT gene to 293T and hUVEC, and hTERT mRNA and protein expression were remarkably increased in infected cells. Conclusion: The study successfully constructed recombinant plasmid pCDH-hTERT, the recombinant lentiviruses can deliver target gene hTERT and have high infection efficiency. Extrinsic hTERT gene can reconstruct telomerase and settle a basis for establishing immortalized fibroblast of cells.
Objective: In order to establish miR-122 transgenic mouse with GFP, two construction strategies of miR-122 overexpression plasmid were compared. Methods: The pre-miR-122 sequence (291bp) was respectively cloned into the intron upstream or 3'UTR downstream the GFP gene,which was inserted into pBROAD3 vector before. After the two plasmids were transfected into 293T cells, the mRNA expression levels of miR-122 and GFP were detected by Q-PCR, and the fluorescent of GFP in 293T were observed by fluorescence microscope. Another vector, miR-122 sensor reporter, was constructed with three repeated antisense sequences of miR-122 inserted into the 3'UTR of luciferase in psiCHECK2 vector. The regulation function of miR-122 was identified by detecting luciferase activity after the sensor reporter and miR-122 overexpression plasmids were co-transfected into 293T cells. Results: These two construction strategies were proved to increased the expression level of miR-122, but only the plasmid with miR-122 in GFP 3'UTR could express activated GFP. Conclusion: Inserting microRNA into 3'UTR of GFP will not influence both of the expression level and the activity of miR-122 and GFP. The vector pBROAD-GFP-miR-122 is adapted for the study of transgenic mouse.
Since the discovery of RNA interference (RNAi), RNAi has become an important method for studying genes’ function in mammalian cells. RNAi-mediated knockdown or post-transcriptional gene silencing of the gene of interest is achieved either by transfection of synthetic small interfering RNAs (siRNAs) or by expression of short hairpin RNAs (shRNAs). However, if the targeted gene is essential for cell growth and viability, constitutive expression of shRNAs may not be possible or appropriate. Doxycycline-induced gene silencing, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying these genes’ function. Conventional method is to construct binary system to perform inducible gene silencing, including inducible RNA polymeraseⅢ( polⅢ) promoters with embedded tetracycline operators (tetO) and co-expression of the tetracycline regulatory protein, TetR. In the absence of tetracycline or its derivative doxycycline, TetR homodimer binds tightly to the Tet operon(s) inserted within the promoter and acts to suppress transcription. Upon its addition, tetracycline binds with high affinity to the Tet repressor homodimer and causes a conformational change in the repressor that renders it unable to bind to tetO, allowing expression of the shRNA transcript. However, the process for generate stable cell line whose target gene knock-down is time-consuming multi-step process which limited transgenic animals’ expansion. In addition, most shRNA expression units rely on polⅢ promoters such as the H1 or U6 promoter, but it is better to use RNA polymeraseⅡ( polⅡ) promoters for tissue specific gene silencing. To overcome the limitation of shRNA expression systems in mammalian cells, a single vector which only incorporates tetO-hUbc promoter, tTS was used in this study. Firstly, five pGenesil1.0-shRNA plasmids were constructed and selected for the most effective shRNA target on porcine Nanog by Realtime-PCR. In order to generate a single-vector harboring polⅢ promoter allowing for rapid generation of stable cell lines for regulatable shRNA expression targets on porcine Nanog, the selected effective shRNA whose interference efficiency could reach 80% was inserted to a modified doxycycline-induced gene silencing vector TREsilencer. Realtime-PCR analysis demonstrates that efficiency of Nanog silencing could reach about 70%, with increasing concentration of doxycyline. It is a fundamental work for generating Nanog stably silenced porcine cell lines and further studying on Nanog genes’ function.
Vascular endothelial growth factor (VEGF) plays a key role in physiological and pathological angiogenesis and is a potent and critical target as blocking tumor growth and metastasis. The process described in this report involves a complex auto-induced expression in Escherichia coli and a downstream purification process consisting of protein refolding and three chromatography steps in order to obtain the functional rhVEGF165. Biological activity of the purified 38kDa homodimer was verified by the induction of the proliferation of human umbilical vein endothelial cells (HUVECs). The EC50 for this effect was 2.4ng/ml. Finally, three Anti-VEGF hybridoma cell lines were obtained after immunization, fusion and preliminary screening.
KLF4 is one member of the kruppel-like factors (KLF) family, which is an important transcription factor to maintain the pluripotency of embryonic stem cells (ESCs). Protein transduction domain (PTD) can carry macromolecules into cells. In order to obtain the recombinant KLF4 protein with penetrating membrane activity,the KLF4 gene was expressed as a fusion protein PTD-KLF4 combined with PTD using prokaryotic expression vector PKYB. The fusion protein PTD-KLF4 was purified by Ni affinity chromatography and identified by Western blotting analysis. The fluorescein isothiocyanate (FITC) labeled PTD-KLF4 was used to investigate the penetrating ability of the fusion protein into Chinese hamster ovary (CHO) cells. And the bioactivity of PTD-KLF4 binding the target DNA sequence was detected by fluorescence resonance energy transfer (FRET). The results showed that the recombinant PTD-KLF4 successfully entered the cells and located in the nucleus with the transmembrane efficiency of (22.29±2.1)%.The recombinant PTD-KLF4 caused cell shape changes and had specific binding capacity with its target DNA sequence.The preparation of the recombinant PTD-KLF4 laid a good foundation for the induction of pluripotent stem cells (IPSCs) with exogenous protein.
On the basis of TB fermentation medium, an optimized fermentation strategy of cutinase-CBM by E.coli was developed through single factor analyzing and orthogonal design, which can be illustrated as follows: glycerol 5g/L, peptone 16 g/L, MgSO4·7H2O 2.5 mmol/L, K2HPO4 13.7 g/L, KH2PO4 1.53 g/L, 1 g/L lactose and 0.75 g/L glycine (final concentration) were added in the prometaphase of logarithmic phase of growth curve, and the fermentation period lasted 48h under 30℃. An extracellular enzyme activity of 63 U/ml was reached, about 3 times that of the control (20 U/ml). Further efforts were made to investigate the influence of other factors, such as heat-shock, osmotic agent, and temperature, on the secretional expression of cutinase-CBM. It is found that with the addition of 75 mmol/L L-proline, heat shocked for 1h at 37℃ or 0.5h at 47℃, then shifted to 25℃, the final extracellular production of cutinase-CBM can reach 90U/ml, 4 times that of the control cultivated at constant temperature.
Study on the optimization of B.subtilis H1 and A.niger P1 mixing fermentation to degrade the macromolecular protein of cottonseed meal was carried out. The results showed that the optimal conditions were as follows: cottonseed meal 40 g, (NH4)2SO4 0.2%, bran 15%, water content 50%, nature pH 6.0, temperature 30 ℃, the optimal time 60 h, the optimal inoculation concentration was 15% and the optimal inoculation ratio 2 ∶ 1 (A.niger P1 ∶ B.subtilis H1). Under the optimal level determined, the content of small peptide increased to 18.36%, the length of average peptide chain decreased to 4.23 and the digestibility of protein increased to 88.59%.A.niger P1 can produce rich acid protease, while B.subtilis H1 can produce rich neutral and alkali protease. Under the combined fermentation, the enzyme activity significantly increased. The peptides composed of more than 10 amino acids in the water-soluble peptides were almost degraded to peptides composed of 1~3 amino acids. In short, combined fermentation was better than single fermentation. The two strains have synergistic effect.
Taking co-fermentation both of Bacillus subtilis D17 and Bs16 (1:1) as based drugs, through the compound design, the preparation stability, resistant, the indoor outside antibacterial bioactivities experiment, carrying out best emulsion screening. The result shows that: the toxicity of emulsion 1 and emulsion 9 was the strongest. It has the high control efficiency to the grey mold. It may also reach above 85% in the plot experiment to the grey mold's against effect. Its control efficiency is similar with the common chemistry agricultural chemicals. Its effective date which lengthens to 15 days above has the significance difference with the common chemistry agricultural chemicals. Its anti-ultraviolet ability, passed through 180 minute ultraviolet illumination preparation unit volume the effective live fungus quantity still to be possible to reach above 109cfu/ml, is very strong. The storage stability is good, the normal temperature seal evades under the cloudy condition to guarantee the nature time to be possible to amount to above one year. It would be the new green environmental protection biology agricultural chemicals which has the high production and the commodity development value.
With the widely use of MAbs and their fragments, traditional hybridoma technology cannot meet the increasing demands of Mabs or antibody fragments. Owing to the rapid development and matures of recombinant DNA technology and biological engineering Technology, it is possible to manufacture Mabs/fragments in large scale. Firstly, the most popularly expression system such as Escherichia coli (E.coli ), yeast, insect cell and mammalian cell system were listed and compared. To achieve high-level expression, secondly, the strategies including modification of recombinant protein (fusion of protein, site-specific mutagenesis), expression in vivo, choice of expression system, optimization of codon and expression circumstance were discussed. Finally, the prospective of the field of antibody/antibody fragment expression was described with consideration of bioinformatics playing an important role in this area, and it is the right time to construct the platform of high expression.
The directed evolution of wild-type catalase from Cervus nippon was studied, which mimicked evolution on a laboratory timescale. The mutant library of catalase gene was constructed using error-prone PCR and DNA shuffling, and its sequencing result showed mutant rate to be 0.329% and 27.58%. The mutant rate using error-prone PCR was 10 fold higher than normal PCR, while the mutant rate using DNA shuffling was much more higher. However, tremendous mutant rate may affect or even disable gene transcription and translation. A suitable mutant rate is needed to guarantee fluent gene expression. Furthermore, both wild-type and mutant catalase sequences selected randomly were analysed to obtain their Neighbor-Joining phylogenetic trees.
Nanotechnology is an emerging technology with enormous potential in biology and biotechnology, medicine and medical technology. Its application in biomedical sciences presents many revolutionary opportunities in the fight against all kinds of cancer, infection and other diseases. The wide variety of core materials coupled with tunable surface properties such as optical, electronic and magnetic properties, making nanoparticles an excellent platform for a broad range of biological and biomedical applications. Several common nanomaterials including gold nanoparticles, quantum dots, magnetic nanoparticles, carbon nanotubes and silicon nanowires in the application of proteins, DNA, metal ions and biologically relevant molecules detection are reviewed.
β-catenin, encoded by CTNNB1 gene on chromosome 3p21—22, is a multi-functional protein which mediates cell adhesion and signal transduction. It plays an important role in embryonic development and tumorigenesis by participating in regulation of cell proliferation and differentiation. In recent years, an increasing number of studies have revealed that inappropriate activation of the canonical Wnt pathway plays an important role in the pathophysiological process of hepatocellular carcinoma (HCC). As a pivotal molecule in Wnt signal transduction pathway, the abnormal expression of β-catenin is closely associated with the development of hepatocellular carcinoma. The latest progress of the role of β-catenin in hepatocellular carcinoma is reviewed.
Rheumatoid arthrisits(RA) is a systemic autoimmune disease that exact pathogeny is unknown. Generally speaking, the pathogenic factors of RA are due to environmental factors and genetic factors. In genetic factors histocompatibility leukocyte antigen(HLA) is one of the most important elements. In addition, peptidylarginine deiminase 4 (PADI4) which does not belong to HLA has been thought to participate in the development of RA recently. PADI4 can catalyse peptidylarginine to citrulline in the presence of Ca2+. This action leads to posttranslational modification of protein. Citrullination brings to conformation change in peptide chains and active sites transformation. In various population, the relation between the SNP which located in PADI4 gene and pothogenesy of RA is different. PADI4 is up-regulated in RA patient’s blood serum and results in autoimmune antibody. PADI4 also citrullinates various protein induced autoimmune response which contributes to the development of RA. Lately other reports show that PADI4 is also relative to tumors, ulcerative colitis as well as multiple sclerosis. Although researches about PADI4 have been made a lot of significant progresses, abundant of unknow have always been existing.
Marine microalgae have proved to be an important and promising resource for antibiotics for their special growing environment. The progress of screening antimicrobial components from marine microalgae and the methods used in the screening were reviewed. Also, development on new screening strategies and the prospects and trends for future screening were briefly introduced.
The reactions catalyzed by nitrile hydratase have been widely applied in organic synthesis. As an important class of catalytic agent, NHase (nitrile hydratase EC4.2.1.84) can converts nitriles to the corresponding acidamide. Because of their inherent enantio-and regioselectivities and other benefits, NHases are attractive as green, mild, and selective catalysts for setting stereogenic centers in fine-chemical synthesis and enantiospecific synthesis of a variety of acrylamides. The literature has been surveyed to provide a comprehensive coverage of the study of NHases in China at present including biodiversity of strain-producing NHase, biochemical and structural properties of NHase, strain improvement, as well as construction of gene engineering strains. Literature has also been cited to describe methods of immobilized cells and application of nitrile hydratase.
Greenhouse effect, water resource crisis and energy crisis are the three major challenges in front of human beings in the 21st century. Microalgae, as a kind of aquatic plant, have become a hot global research topic in the field of CO2 emission reduction, wastewater ecological recovery and biological energy. The application research progress in using microalgae in the field of CO2 biological trapping and wastewater ecological recovery is summarized. Based on microalgal biological processes research concentrating on optimum design of one unit and lacking of fully cognition toward the importance of interconnection, The conception of coupling these factors are putted forward. The new biofuel strategy is biodiesel production of high oil content microalgae coupled with wastewater treatment and flue gas, then algal residue and coal both vaporize. Efficient global optimization and environmental comprehensive control is important. To develop low carbon economy is an effective way in China, The future research emphasis and prospect of microalgae interconnection are also mentioned.
Stem cell has become the global focus for its promising potential in research and practice. The governments, Institutes and enterprises all over the world have been increasing investment, and have made great progresses. Stem cell related papers and patents have sharply going up since 2001, and researches on clinic and pre-clinic trials became more and more though majority still focus on umbilical cord blood stem cells. USA remains the top position in all aspects in stem cell field. The numbers of stem cell related papers and patents produced by China are ranking top 10 around the world, but no Institute belongs to top one, and not many new products are developed successfully. China has to learn how to actively react to the changes resulted from related looser policy in USA.
According to the characteristics of biomedical industry and Porter's Diamond model, forming a six factors diamond model.Taking Shijiazhuang as example, analysis of six elements which affecting the industrial competitiveness,such as production factors, demand, related and supporting industries elements, structure and competition elements of corporate strategy, government, innovation. And illustrates the path of enhancing its competitiveness, such as select the appropriate cluster development model based on comparative advantage and local conditions, actively promote the combination of government, industry, academia, research and medical, through various means to improve innovation, actively undertake the outsourcing services of biomedical industry, strengthen the agency and information to support the construction,improve the social and cultural support for the biomedical industry, etc., in order to ensure the Shijiazhuang sustainable development of biomedical industry.
