A specific promoter in hepatocytes--hAATp (human α1-antitrypsin promoter) and a HCR (hepatic control region) which can function as an enhancer using PCR were successfully obtained. On basis of this, several kinds of chimeric hepatocyte specific hAAT promoter with different amount of HCR enhancers were constructed. Then luciferase reporter gene was inserted into the downstream of the chimeric promoters. And the promoter activities of these different promoters and their specificity in hepatocytes were analyzed by detecting the expression activity of luciferase in different recombinant plasmid separately based on the transfection of the recombinant plasmids into HepG2 cells, Hepa1-6 cells, HEK293 cells and U87-MG cells. The result showed that among these reconstructed chimeric promoters, the one carrying three enhancer possess the best specificity in hepatocytes. These results lay the foundation for target gene therapy in human liver diseases.
Pichia Pastoris is one of the most successful heterologous protein expression systems until now. It doesn't have the endotoxic problem which always existed in the prokaryotic expression system nor the viral contamination of recombinant proteins produced in mammalian cells culture. Furthermore, unlike bacteria, yeasts can carry out the similar post-translational protein processing as higher eukaryotes, such as signal peptide cleavage, disulfide bonds formation and the glycosylation. However, it is not clear which protease may present in the culture media and degrade heterologous protein which leads poor yield of protein of interest. Objective:Comparing the GS115 proteomics differences cultured in different carbon source media to optimize the heterologous protien expression system.Methods:By using LC-ESI- MS/MS combined with Griffin's new calculation method, intracellular and supernatant protein profiling and protein abundance of GS115 in 4 different carbon source media were identified and compared.Results:Intracellular and supernatant protein profiling and related protein abundance of GS115 in 4 different carbon source media were obtained with LC-ESI-MS/MS analysis and Label-free normalized quantification method. Conclusions:The findings will be helpful to develop novel strains which may optimize the Pichia pastoris heterologous protein expression system.
Preparation of Nano elemental selenium (Nano-Se) bio-transformed from Se enriched S. platensis (Se-SP) and scavenging activity on oxygen free redicals was investigated. Nano-Se was harvested from high cells density cultures of Se-SP with total Se supplementation of 600 μg/ml in form of sodium selenite. The shape and size of Nano-Se was characterized by atomic force microscope (AFM), transmission electron microcope (TEM) and energy-dispersive X-ray (EDX). Se contents were detected by inductively coupled plasma mass spectrometry (ICP-MS). The scavenging activities of Nano-Se on superoxide anions and hydroxyl radicals were detected by chemiluminescence method. The data showed that the bio-transformed Nano-Se was constructed mainly by elemental Se. A 73% fraction of Nano-Se was collected by gradient centrifugation, in which it was spherical in shape and uniform in size with average diameter of (61±17)nm. In vitro maximum scavenging rates of Nano-Se on superoxide anions and hydroxyl radicals were 30.1% and 27.6%, and the correspondence EC50 were 0.8 and 2.2 μg/ml, respectively. The scavenging activities of Nano-Se on oxygen free radicals were much higher than that of selenomethinoine and other Se containing compounds isolated from Se-SP at the same dosages. In conclusion, present results suggested that Nano-Se produced by high cells density cultures of Se-SP is a novel Se species with anti-oxidative activity in vitro.
In order to investigate gibberellin-producing capacity in the continuous fermentation, the cells of Gibberella fujikuroi were immobilized in the form of Ca2+-alginate beads. The conditions for immobilization were investigated. Then, the growing capacity and production potential were discussed in the immobilization beads containing different age mycelium at a different concentration of glucose. The experimental results showed that the optimal immobilization condition was 3 g/L sodium alginate and 8 g/L cell in the presence of 3mol/L CaCl2. The maximum specific gibberellin-produced rate was 4.61×10-3/h as the culture condition was Ca2+-alginate beads immobilized 90 h age hypha and 2 g/L glucose concentration in shake-flask cultures, while the mycelium growth basically stopped in immobilized beads. In this condition, the specific production rate of gibberellic acid was 4.82×10-3/h in a continuous fixed-bed bioreactor by immobilized mycelium of Gibberella fujikuroi, was 1.87 times than the maximum specific production rate in the batch fermentation.
A ripid and efficient method for separation of unsaturated fatty acid is described as the FOLCH method that is extract total lipids of blood plasma with 1∶20 blood plasma to extrcatant(methanol∶chloroform=1∶2). The method is modification from it that allows the proption of blood plasma to extrcatant(methanol∶dichloromethane=1∶2)reducing from1∶20 to 1∶5. we prepared silver nitrate-silica gel by the Ghosh method, and, drew the adsorption isotherm of linoleic acid to understand the character of silver nitrate-silica gel for adsorbing unsaturated fatty acid. In addition, fatty acids were eluted with a mixture of n-hexane∶dichloromethane∶ ethyl ether=89∶10∶1 after the sample has been loaded column. After that, linoleic acid was quantitatively analysed and the others fatty acids were qualitatively analysed by GC and GC-MS. There are distinct differences for purification of linoleic acid and the others fatty acids in the eluent, the silver nitrate-silica gel can well purificated unsaturated fatty acids in blood plasma.
Presently under the Regulation (EC) No 1830/2003, the food and feed produced from genetically modified organisms are mandatory labeled as its GM contents beyond a specified level, threshold. The fluorescence quantitative polymerase chain reaction was used as the standard method to detect the contents of the transgenic component in China and European countries. It is recognized that in order to be able to judge if an analytical results exceeds a threshold; the MU must be estimated and reported together with the measurement result. Nowadays, on the relative quantification of GM products with real time PCR methods, the copy number ratios between endogenous gene and extraneous gene are use to stand for GMO mass fractions (m/m) in food and feed. The procedures were devised to estimate the uncertainty that originates in the analytical processes and use Roundup Ready soybean as an example to demonstrate how to estimate the analytical variability of quantitative analytical results obtained by real-time PCR, basing the data derived from in-house validation of the PCR method.
In order to establish an efficient transformation method for Aspergillus niger, the effect of different conditions on transformation frequency which is based on the unique advantages of Agrobactrium tumefaciens-mediated system(ATMT) were studied. Binary vector, pBI-hph was constructed and transferred into A.tumefaciens LBA4404 by electroporation. Filamentous fungus A.niger TCCC41056 was used as the hose strain and hph gene was used as selective marker, the transformation system of A. niger mediated by A.tumefaciens was established. Besides, the factors affecting the transformation efficiency such as spores freshness,A.tumefaciens'concentration,spore concentration,co-culture temperature and time were analyzed. The result of PCR showed that the T-DNA was integrated into genome of the A.niger. All 8 randomly selected hygromycin B-resistant transformants tested were stable after 10 subcultures onto complete medium without hygromycin. Based on the above researches, the conditions of ATMT were optimized and much higher transformation efficiency was got:about 83 transformants/107 spores. From of numerous transformants, one A.niger mutant strain that high-yield of glucoamylase was screened. ATMT was applied in A.niger and this transformation system will provide a powerful tool for the further study on function genes of A. niger.
The Nile red(NR)fluorescence and Fourier transform infrared micro-spectroscopy (FTIR)was used to determine lipid content over time in freshwater microalgae Scenedesmus obliquus JNU20. The optimum staining conditions for S. obliquus JNU20 were as follows:pretreated 40s with microwave before staining, the volume fraction of dimethylsulfoxide was 1%, the final concentration of Nile red dye was 1.5 μg/ml and incubation 5 min at 40℃. At the different growth phases, fluorescence spectrometer and FTIR spectrometer and gravimetric methods were used to determine lipid content in S. obliquus JNU20 grown in limiting concentration of nitrogen (0.5 g/L NaNO3), the dynamic process of lipid body formation was also observed by laser scanning confocal microscopy, the results of spectrofluorimetric and FTIR spectrometric methods were very strong correlated with the results determined by gravimetric method (R2=0.925 8, R2=0.984 4), respectively. This strong correlations thus validate that the spectrofluorimetric and FTIR spectrometric methods described provide rapid, easy manipulated and reliable methods for quantification of lipids in microalgae. It was the basis of wide-scale screening of oleaginous algal strains and tracking the process of lipid accumulation in oleaginous algae.
Background:Inositol-requiring 1/X-box-binding protein 1 (IRE1/XBP1)-mediated signaling represents the most conserved and important branch of the unfolded protein response. Xbp1 mRNA is spliced by IRE1 and then XBP1s protein with transcription activity will be translated. It has been proved that the activator of XBP1 may be used as therapy to ease type Ⅱ diabetes, regulate adipogenesis, improve neurodegenerative disease and adjust immune system. But to tumor, XBP1s is essential for survival and is required for its growth and inhibition of XBP1 will be discussed as a therapeutic strategy. Objective:To establish a high-throughput screening model targeting the IRE1/XBP1 signaling pathway at the cellular level for discovery of new inhibitors of IRE1/XBP1 pathway. Methods:A new plasmid named pCAX-F-XBP1△DBD-luciferase was constructed. The plasmid and pcDNA3.1 were cotransfected into HEK293 cells and stable lines were selected with G418. Results:First, ER stress inducer tunicamycin (TM) was used to evaluate the sensitivity of stable lines on IRE1/XBP1 pathway. And clone named 6# was chose for further study. Then factors related with the model including density of seeded cell, the final concentration of DMSO and concentration and incubation time of TM were optimized (Z '=0.62). And 449 compounds including multiple kinase inhibitors were screened. Totally 27 positive hits were screened out and proteasome inhibitor MG132, kinase inhibitors Sunitinib and Staurosporine were identified with IC50 of 6.61(±1.51)μmol/L, 6.25(±0.36)μmol/L and 48(±8)nmol/L respectively. Conclusion:a high-throughput screening model targeting the IRE1/XBP1 signaling pathway at the cellular level was established effectively.
DNA labeling with thymidine analogue bromodeoxyuridine (BrdU) is a powerful tool for analyzing DNA replication and repair. But the technique was limited to use for Saccharomyces cerevisiae due to lack of thymidine salvage pathway which promoted BrdU binding to DNA.Two genes, herpes simplex virus thymidine kinase (HSV-TK) and human equilibrative nucleoside transporter 1 (hENT1), were inserted to the yeast genomic DNA by using an integration plasmid p405. The results showed that when the concentration of BrdU was 0.2mg/ml, it can be labeled to S. cerevisiae genomic DNA during the mid-logarithmic period. The fluorescence signal intensity at 3h was stronger than those at 1h or 5h in vitro. And data from detecting in vivo showed that 55.3% of the S. cerevisiae genomic DNA can be integrated with BrdU. These indicated that a new method with BrdU for labeling to S. cerevisiae genomic DNA was established. The protocol can be used easily for analysis of genomic DNA replication and repair for S. cerevisiae.
Fluorescence spectroscopy, UV spectroscopy, Fourier transform infrared spectroscopy(FTIR) and Far UV circular dichroism (Far-UV CD) were done to characterize the structural changes of papain influenced by heavy metal ion Hg2+. And the denaturation and inhibition mechanisms of papain under the action of Hg2+were studied. The results indicated that Hg2+ was a strong inhibitor of papain. 90% enzymatic activity was lost at 10－4mol/L Hg2 +concentration. With Hg2+concentration increasing, the secondary structure and microenvironment of papain changed obviously. (α-helix + β-sheet) reduced, the content of random coil increased, and the secondary structure of the enzyme structure turned from regulation into disorder and random.
Human respiratory syncytial virus (RSV) is the leading viral agent of serious respiratory disease in infants and young children worldwide, infecting nearly all children one or more times by age 2. An intranasal live vector-based RSV vaccine mimics natural infection and presumably synthetized proteins in conformation similar these synthetized by natural infection. An intranasal live vector-based RSV vaccine is able to escape the immunosuppressive effects of maternal serum antibodies and avoid the immune-mediated enhanced disease. The advances in the live vector-based RSV vaccine is reviewed.
Among viral vectors used in gene therapy approaches, recombinant adeno-associated virus (rAAV) vectors have shown great promise because of their efficacy and safety profiles. However, application of rAAV in gene therapy is limited by its packaging capacity of about 5.0 kb. Recently, due to a better understanding of the basic transduction biology of rAAV, several novel strategies have been developed to breakthrough the gene size limit of the small viral vector such as trans-splicing and homologous recombination techniques. The package limit property of rAAV can also be used to reduce the contamination of the replication-competent AAV in large scaled production process in order to meet the safety requirement of clinic trials.
Gene-targeting efficiency in the land plant Physcomitrella patens (Bryophyta) can only be compared with that observed in Saccharomyces cerevisiae. Physcomitrella patens, as the new "green yeast", might well become a major tool for functional genomic studies of multicellular eukaryotes. In addition, the relatively simple developmental pattern and the haploid gametophyte in the life history make it a suitable genetic tool. Molecular tools and genetic information are rapidly developing for P. patens. The current knowledge of Physcomitrella patens transformation system including the construction of vector, the transformation method and the preparation of the host cells are reviewed. The application of the Physcomitrella patens genetic transformation system was exampled at the last.
As a milestone in the development of DNA sequencing, high-throughput sequencing technology provides an unprecedented opportunity for the modern life sciences. The recent progress on this technology, including the second generation sequencing technology (represented by 454, Solexa and SOLiD), the third generation sequencing technology (represented by HeliScope TIRM and Pacific Biosciences SMART)and the Ion Personal Genome Machine sequencing technology are summarized. Then, the application of the high -throughput sequencing technology in genome sequencing, transcriptome sequencing, gene expression regulation, detection of binding locations for transcription factors and methylation analysis are summarized. Finally, the disadvantages and the prospects of this technology were discussed.
Advanced biofuels are high-energy liquid transportation fuels derived from sustainable biomass feedstocks including algae, which has attracted attention during the past few years as renewable and environmentally friendly alternative fuels, but many obstacles still remain in technical development and demonstration. In recent years, there has been a lot of breakthrough and progress in synthetic biology area, and therefore synthetic biological research has shown promising prospects in many areas, especially in that of advanced biofuels. The development of advanced biofuels and synthetic biology and its role in biofuels research and development is thoroughly demonstrated, and the opportunities and challenges of synthetic biology are also discussed.
A brief analysis about National High Technology Research and Development Program(863)drug design based on function genomes & structure genomes topics in "Eleventh Five-Year Plan" was given. The details about topics plan, institutions carrying out the topics and representative research results were presented, these informations maybe be useful to scientific and technical workers.
Based on the data released by National Agricultural Statistics Service(NASS) of United States Department of Agriculture(USDA), First introduces the planting changes of major crops in U.S., then the planted acres and the varieties of major genetically engineered (GE) crops in U.S. in 2000-2011 was analyzed, in order to summarize the status of major genetically engineered crops in the U.S.. Finally introduces the development of corresponding GE crops in China and got some inspiration for China.
