The structural study of effector protein is important for studying thepathogenesis of legionella pneumophila. A construct for plasmid of LepB-PFastBac1-GST and 9 constructs for plasmids of LepB-truncation-PGEX-1-GST were successfully constructed and induced to express in insect cell or bacteria Bl21 (DE3)，the obtained recombined protein was purified and crystallized by the vapor diffusion method. The crystal of the trunction of LepB -480-679 grows. The results Provide a basis for helping resolve the structure of LepB and further research work on the pathogenesis of legionella pneumophila.
Polymerase chain reaction (PCR) for DNA and RNA amplifications in vitro has a profound impact on modern molecular biology. Since design of proper primers is crucial for the success in PCR, many parameters have been used for primer design. However, the effect of DNA polymerase binding to primer/template duplex on PCR efficiency was not taken into account in conventional primer design programs. To reveal whether or not the DNA polymerase-primer/template binding affects the PCR efficiency, here we built structural models for the Taq DNA polymerase in complex with different primer/template sequences, and designed PCR primers according to relative binding free energies calculated by MM/GBSA method. We verified our primer design approach using Human Serum Albumin (HSA) gene and Mycobacterium tuberculosis pyrF gene, and found that the PCR efficiencies of different designed primers for both tested genes correlated well with the calculated binding free energies. Our finding indicates clearly that the DNA polymerase-primer/template binding affects the PCR efficiency significantly. Thus, the calculated binding free energy could be used as a new parameter to design efficient PCR primers.
FGF2 is one of the most important factors in tumor angiogenesis. Blocking FGF-2/FGFR activity with antibodies could be proposed as a potential therapeutic strategy, especially in the treatment of tumors. A mouse anti-FGF2 monoclonal antibody (mAb), designated as 9B9, has previously been produced and characterized by our laboratory. We report here the construction and the optimized expression of mouse-human chimeric antibody derived from the mAb. cDNAs encoding variable regions of heavy and light chains were prepared from 9B9 cells by polymerase chain reaction, and introduced to eukaryotic expression vectorsp Lexm containing cDNA for human gamma1 constant regions, respectively. Cotransfection of the vectors into HEK 293T cells resulted in production of antibody and detected by western blots of reduced and non-reduced SDS-PAGE. The chimeric antibody has a special binding activity to FGF2 and can inhibit the binding of FGFR1βⅢc to immobilized FGF2, (IC50=6.25μg/ml) in ELISA. On the other hand, we optimized the recombinant plasmids through woodchuck hepatitis virus post-transcriptional regulation element (WPRE) and acidic Fibroblast Growth Factor (aFGF) to improve chimeric antibody yelid. The results suggest that WPRE enhanced expression of the recombinant antibody, while aFGF can not effectively promote the expression of chimeric antibody, and with the increase of aFGF, antibody expression quantity reduces instead. Furthermore, recombinant antibody yield was enhanced 12- to 18-fold compared to levels at 37 ℃ by exposure of cells to mild hypothermia (31℃ to 33℃) after transfection. This simple and efficient approach of antibody production is expected to provide a sufficient amount of antibody for screening experiments.
In order to obtain a strain with good genetic stability and high activity of secondary metabolites, the wild-type strain Gibberella moniliformis EZG0807 was mutated by a large gradient superconducting magnet (JMT-16T50F) which can simulate micro and hyper-gravity environment. After G. moniliformis EZG0807 was mutated for 24 h, 48 h and 72 h under these conditions, 139 mutants were isolated with the spread plate method. Among them, a highly active mutant M7226 was selected according to the ability of antibacterial and antitumor activity with the filter paper and MTT method, respectively. Furthermore, the biomass of mycelia growth and the bioactivity of secondary metabolites of the strain M7226 within ten generations were investigated to detect its genetic stability. Accordingly, a highly active and stable heritable strain M7226 was got after mutagenesis with the superconducting magnet, which demonstrated that this superconducting magnet can be used as a new method for mutagenesis breeding and laid the foundation for the subsequent separation and purification of secondary metabolites.
Flg22 is a highly conserved amino acid sequence in the N terminal of bacterial flagellum protein, which can induce plant natural immune response. In order to fully understand systemic responses of plant to bacterial pathogens, two sequencing libraries of Arabidopsis seedlings treated and not treated with flg22 were constructed and sequenced using Illumina Hiseq2000. Comparison of the two samples showed 1 200 differentially expressed genes (DEGs), including 290 down-regulated and 910 up-regulated genes. GO functional enrichment and KEGG pathway enrichment analysis revealed that the DEGs were associated with energy metabolism, amino acid metabolism and biosynthesis of secondary metabolites. Glucosinolates are important secondary metabolites involved in innate immune response against pathogen, therefore it is of necessary to analyze glucosinolates metabolism pathway. After flg22 treatment, genes involved in indolic glucosinolate biosynthesis pathway were up-regulated significantly，which is further demonstrated by Real Time RT-PCR, while aliphatic glucosinolate pathway almost had no change, indicating the important role of indolic glucosinolates in plant defense responses. This research provided numerous genetic data to deeply understand pathogen induced defense and the contribution of indolic glucosinolates.
Recombinant human interleukin- 12 (rhIL-12) is a heterodimeric glycoprotein that has been used to treat diseases such as tumor, parasites, viral infections and hematopoietic disorders. For the structure confirmation is important for quality control, in this paper, the disulfide linkage, N-glycosylation sites and C-terminal amino acid sequences of rhIL-12 expressed by CHO cells were analyzed, and the rhIL-12 underwent enzymolysis using three non-reducing enzymes: Trypsin, Chymotrypsin and Glu-C, to break between cysteine residues and form the disulfide-linked peptides, and then peptide samples were analyzed using LC-MS/MS to identify seven pairs of disulfide bonds presenting in rhIL-12 sample that match the theoretical conditions. After reduction of disulfide bond and alkylation modification protection, the rhIL-12 underwent enzymolysis using Trypsin, Chymotrypsin and GluC, and mass spectrum peptide mapping and C-terminal amino acid sequence analysis of the peptide segments were then carried out using LC-MS/MS, to identify eight amino acids at p35 subunit C-terminal and 15 amino acids at p40subunit C-terminal of rhIL-12. After reduction and alkylation, the rhIL-12 samples were degenerated and enzymolysised, and the enzyme-digested products of peptide segments were thereafter treated with PNGase F in H2O and H218O respectively. Through tandem mass spectrometry analysis of the change in molecular weight of peptide fragments, three N glycosylation sites of rhIL-12 were exactly identified, which were site 71 and site85 of p35 subunit, and site 200 of p40 subunit. Through establishing the method combining enzymolysis with mass spectrometry identification, ultimately it was demonstrated that disulfide bond site, C-terminal amino acid sequence and glycosylation sites of new drug rhIL-12 were consistent with the theoretical conditions.
Influence of different fermentation conditions of Chryseobacterium indologenes on its protein-glutaminase producing ability, the extraction and application of enzyme were studied. The optimal fermentation condition was concluded from the studies of seed culture profile, fermentation temperature, rotation speed, volume of medium, carbon source and nitrogen source. The result showed that the optimal fermentation condition was 30℃, 200r/min, 250ml shake flask with 25ml culture medium, sucrose as carbon source, poly-peptone as nitrogen source, fermenting for 10~12h. The study of preliminary extraction indicated that when the enzyme solution was 4-fold concentrated and precipitation with 4 times volume of ethanol, the maximum of total enzyme recovery of this two steps were both obtained, were 84.99% and 76.07%, respectively. When casein was incubated with the enzyme for 2h, deamidation degree of deamidated casein could reach 41.03%. After the incubation time of 24h, deamidation degree was no longer increase, and the solubility of casein improved.
Objective：To express recombinant ES-Kringle5 fusion protein in prokaryotic cells, purify and identify the bioactivity of expressed product. Method：The nucleotide sequence encoding the 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin and Kringle 5 via a peptide linker were synthesized and inserted to pMD18-T, then the sequence cloned into vector pET25b. And this recombinant plasmid was transformed to E.coli BL21(DE3) for expression by lactose induction. The expression product was purified by Ni-NTA. The abilities of ES-Kringle 5 to inhibit the proliferation of HUVECs was used for its biological activity assay. Results: The ES-Kringle5 coding sequence was correctly cloned into pET-25b vector. Use lactose and lower the induction temperature can increase the expression level and soluble protein expression. The recombinant protein reached a purity of more than 95% after purification. Bioactivity assay result shows that ES-Kringle5 could inhibit the proliferation of HUVECs. Conclusion：The recombinant ES-Kringle5 fusion protein was successfully expressed in E.coli with high biological activity, which lay the material foundation for its pharmacology study in vivo.
To obtain wild mushrooms having broad-spectrum antimicrobial activity and enhance the antimicrobial compounds productivity were our purpose. The active strains were screened in terms of the methods of well diffusion assay and hypha growth rate. the fermentation parameters for antimicrobial compounds productivity were optimized by response surface analysis and orthogonal test. One white-rot fungus 0331 with broad-spectrum antagonistic to pathogens was obtained, and it was identified as Cerrena sp. by ITS-5.8 rDNA sequences analysis. The fermentation broth showed the antimicrobial activity against Staphylococcus aureus, Escherichia coli, Monilia albican, Bacillus subtilis and Rhizoctnia solani. The optimal medium for 0331 was composed of (in g/L): potatoes 13.99, wheat bran 7.00, Sucrose 41.58, KH2PO4 2.00, MgSO4·7H2O 2.00, VB1 0.027. The optimal culture conditions were as follows: the initial pH value of medium 5.0, the fermentation period 10 d, packing volume 110ml/250ml, inoculation 8mL/100ml, rotate speed 180r/min, temperature 28℃, respectively. The 0331 strain showed obvious antimicrobial activity, with an increase of 30.37% compared to the original fermentation parameters.
Due to fungal extracellular polysaccharide have high adsorption and high viscous characteristics, it is one of the difficulties of extracting the high purity of nucleic acids from extracellular polysaccharide germs. The production of scleroglucan strain Sclerotium rolfsii as the material, obtained high quality genomic DNA by the improved Cetyltrimethyl Ammonium Bromide (CTAB) method. By reducing the production of polysaccharide in the cell culture process to obtain high mycelium, relatively. And used the high concentration KAC and absolute ethyl alcohol to precipitate the crude polysaccharide, then the CTAB/NaCl solution to remove out the polysaccharide. Compared with the gDNA Kits and traditional method, highly yield and purity of gDNA obtained with simple steps. And the product is suitable for basic molecular experiments, such as building genome libraries and PCR.
Highly active antiretroviral therapy (HAART) can effectively suppress human immunodeficiency virus type 1 (HIV-1) replication and plasma viral load, delay the onset, and improve the life quality and survival time. But interruption of HAART leads to the rapid rebound of plasma viral load. Infected cells harboring HIV-1 proviral DNA, mainly resting memory CD4+T cells, are the obstacle for eradication. The transcriptional activation or suppression state determines the infected cells into productive infection or latent infection. This review discusses the intricate mechanism of the HIV-1 transcriptional regulation, such as the integration site and transcriptional interference, cellular transcription factors interacting with HIV-1 promoter to recruit RNA polymerase, epigenetic regulation of transcription, and trans-activating factor Tat and its associated proteins to promote transcriptional elongation.
Stimuli-responsive peptides are referred as "smart" peptides capable of self-assembling into supramolecular structures that are responsive to environment changes. Such kinds of smart peptides can self-assemble spontaneously, so they are also called self-assembling peptides. Based on their property, smart peptides can be used as building blocks to construct materials possessing different conformations and biological functions. In this review, we presented the self-assembly mechanism of smart peptides,with a focus on the great potentials of smart peptides in many biological fields, such as biomedical engineering,as well as in energy applications,and bioseparation engineering.
Fuel ethanol as excellent renewable liquid fuel, has received widespread attention for its exceptional performance. Microalgae is one of the best biomass resources with high photosynthetic efficiency and high biomass yield. Microalgae rich in starch, polysaccharides, cellulose(Iα)and other substances can be used as excellent feedstock for fuel ethanol production with traditional ethanol production technology after simple treatment. Fuel ethanol production with microalgae can alleviate China’s growing energy problems, and reduce greenhouse gas emissions and environmental pollution. The recent research progress on microalgae ethanol reviewed and proposed its developmental potential.
L-serine and L-cysteine have broad applications in food, medicine, cosmetic industry, and so on, the precursor of which are both 3-phosphoglycerate in plants and microorganisms. Bio-based production of these two amino acids reveals bright prospects owing to the benefit of society, economy and environment. Focusing on the achievements in the microbial production of L-serine and L-cysteine, this review summarizes the hot spots of the researchers'attentions nowadays，which can be divided into four parts：the metabolic pathways and their regulation, export and transport systems of relative amino acids，production strains and metabolic engineered strains for producing L-serine and L-cysteine，development of new strains for L-serine and L-cysteine production. Finally, perspectives for developing serine-cysteine overproducers are suggested in view of the going forward of biotechnology.
Since the first biopharmaceutical—"recombinant human insulin" was licensed by US FDA in 1982, and "recombinant human interferon α1b", the first biopharmaceutical in China was approved in 1989, biopharmaceutical industry has become the fastest growing, most dynamic and technology-intensive field. Good manufacture practice and quality control are key contributions to the safety and efficacy of these drugs. General requirements for the control of monoclonal antibodies are embodied in European and United States Pharmacopeia. Currently there are 12 product categories and 34 monographs embodied in Chinese Pharmacopeia volumn Ⅲ edition 2010. In the coming new 2015 edition, a general monograph of products prepared by recombinant DNA technology is intend to be drafted and included under the directions to further improve drug safety as well as to upgrade the technology level of quality control. This paper is to pave the way for the drafting of monograph of recombinant DNA products from the aspects including scope, manufacture and release control etc.
China currently faces the food security problem that demand for food continues to increase while increasing rate of grain yield slows down obviously. Exploring innovation of new technology and methods in crop breeding is a key point to achieve a major breakthrough in breeding of new crop breeding, promote substantial and steady increase of grain yield and ensure china's food security. To achieve these goals, a new thought and research model could be provided by exploring a integrated multidisciplinary transagricultural research mode combined with the traditional theories and methods on agricultural research.‘Transagriculture’is extension and development of modern agriculture. It means a series of research models and solutions in the context of global climate change, resource scarcity and food security, which integrate advantages in various disciplines of politics, economy, science & technology and agriculture, and carry out interdisciplinary, cross-industrial joint research and integrated innovation to solve major problems in agriculture. Rapid development of rice breeding theory, genomics, proteomics, technology of image recognition and processing, automation technology and information technology has provided necessary conditions for such transagricultural research mode. Based on the thought of transagriculture and combined with our joint research project on genotyping instrument for seeds & cloud computing terminal for SSR fingerprint of rice, we analyzed and discussed the research background, basis and technical conditions, feasibility and design scheme of intelligent rice breeding robot, and further prospected its application.
The traditional organ transplantation could not meet the need of the clinical treatment for the restrictions such as organ origin, ethic and body immunity. Tissue engineering therefore come to birth and proceed to grow by responding to successive challenges. At the beginning, the definition of tissue engineering was that a combined utilization of cells with scaffold materials and bioactive factors could be an efficient way to promote the research and application of tissue repair and regeneration. With the deep investigation in this field, the tissue engineering concept was also developed. Modern tissue engineering is an interdisciplinary branch of science that study and develop bioactive artificial substitution to maintain, restore or improve the function of the damaged human tissues by use of the principles of engineering science and life science. Since the birth of more than two decades, the development of tissue engineering has experienced about three stages. Regenerative medicine is an important branch science of modern clinical science, which is closely related with stem cell and tissue engineering. Tissue engineering is a perfect tissue organ regeneration, key research field of regenerative medicine, and main development direction of regenerative medicine. The principles and techniques of regenerative medicine promote the development of tissue engineering.
