Four serotypes of dengue virus (DENV1-4) circulate globally, causing more human illness than any other arthropod-borne virus. So far, licensed vaccine against dengue is not yet available. The greatest risk factor for severe dengue is a previous infection with a different DENV serotype. The theory for why sequential heterotypic infections increases risk of severe disease is "antibody- dependent enhancement" (ADE). Previous research determinants that the predominant immune response is against the E protein which is comprised of three structurally distinct domains (DI, DII, DIII). EDIII might exclusively generate potent neutralizing antibodies with little or no cross-reactivity, and therefore of reduced ability to promote ADE. This suggested that EDIII is more likely to be an attractive vaccine. Here, a tetravalent recombinant dengue domain III protein gene sequence were yeast expression optimized and synthesized, then expression plasmids were constructed and transducted into pichia pastoris to selected an high-efficiency expression strain. EDIII protein were obtained by inducing expression and purification followed by identification using SDS-PAGE, Western blot. The result showed that EDIII expression plasmid was successfully constructed and highly expressed in pichia pastoris. Mice immunized with tetravalent DENV DIII generated higher levels of serum titer. In conclusion, a tetravalent recombinant dengue domain III protein with high purity were obtained. It lays a good foundation for the development of dengue vaccine.
CXCL4,Platelet factor 4 (PF4), belongs to the subfamily of CXC chemokines, which is reported to be involved in numerous biological process by acting on a broad spectrum of different cell types. Here, the study aimed to express and purify recombinant human CXCL4 protein using E. coli expression system and affinity chromotography methods. The results demonstrated a simple and efficient strategy for the production and purification of recombinant human CXCL4 (rhCXCL4) with over 95% purity by gel electrophoresis have been developed. Its biological activity was confirmed in the proliferation-inhibition assay using a tumor cell line, ACHN cells. The rhCXCL4 will facilitate the structural and functional analysis of human CXCL4. The production strategy of rhCXCL4 should have important reference values for the expression and purification of other chemokines.
Objective: To investigate the effect of rhS100A6 on S100A6 mRNA of colorectal carcinoma cell lines HCT116 and SW480, and to explore its possible mechanism. Methods: Recombinant human S100A6 (rhS100A6) protien was prepared by prokaryotic expression. FHC, HCT116 and SW480 cells were treated with 10μg/ml rhS100A6 and recombinant adenoviruses carrying human β-catenin siRNA (Adsiβ-catenin), semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real time polymerase chain reaction (qPCR) was used to detect the expression of S100A6 mRNA, RT-PCR was used to detect the expression of E-cadherin mRNA,Western blot was used to detect the expression of β-catenin of nuclear protein in HCT116 and SW480 cells and immunofluorescence staining was used to detect the distribution of β-catenin. Results: rhS100A6 protein was prepared successfully and significantly increased the expression of S100A6 mRNA and nuclear β-catenin protein of HCT116 and SW480 cells, rhS100A6 treatment promoted β-catenin translocating from cytoplasm to nucleus in HCT116 and SW480 cells, at the same time, the E-cadherin mRNA level was decreased with the treatment of rhS100A6 protein. Compared with rhS100A6 treatment group, the expression level of S100A6 mRNA was decreased in co-treatment group by Adsiβ-catenin and rhS100A6. Conclusion: Extracellular rhS100A6 upregulate the experssion of S100A6 mRNA in colorectal carcinoma cell lines and its mechanism is partly involved in Wnt/β-catenin pathway.
Agrobacterium-mediated transformation were used to construct the transformants of the Curvularia lunata and 1454 transformants were obtained. Then screened eight mutants in which colonial morphology, mycelial growth and the expression of disease related genes changed more distinct. Inoculating the mutants into the detached leaves has been used to screen mutant strains and found that 4 mutant strains whose pathogenicity reduced, and 2 mutant strains whose pathogenicity enhanced. Determining the growth rate, sporulation quantity and the activity of cell wall degradation enzymes showed that the sporulation quantity of the mutants whose pathogenicity reduced were all decreasd and one of the strains did not even produce a spore. The activity of the PG enzyme enhanced generally, but the changes of the Cx enzyme did not have differences apparently. However, the activity of the Cx enzyme of the two mutants whose pathogenicity enhanced was increased.
The 1 477bp promoter of miRNA3026 was cloned, furthermore, four mutant fragments with different length of deletion were constructed and transient expression assay showed that the activity of miRNA3026 promoter is weak. OsTxnDC9 was cloned, the host gene of miRNA3026, which has an open reading frame of 482bp. Bioinformatic analysis indicated that OsTxnDC9 encodes a protein which is highest homology with Brachypodium distachyon. The expression of OsTxnDC9 was highest in one nuclear stage of pollen, which was examined by real-time polymerase chain reaction essay. Subcellular localization suggested that the protein was located in cytoplasm. This research laied the foundation for further exploring the relationship of miRNA3026 promoter and its host gene.
Lepidium apetalum Willd is an important traditional Chinese medicine. Various active components have been extracted from the Lepidium apetalum. However, the genetic basis for their activity is virtually unknown. The transcriptome of Lepidium apetalum was sequenced using the Illumina HiSeqTM 2000 sequencing platform. The clean reads were then de novo assembled into 40 303 unigenes. 27 935 unigene were annotated by a similarity search against SiX public databases. The results showed that 534 genes were assigned to second metabolic pathway. Among them, 4 unigenes were mapped to the glucosinolate, 19 to flavonoids, stilbenoid, diarylheptanoid, 69 to gingerol shikimate biosynthesis pathways, and 92 unigenes were respectively mapped to the phenylalanine metabolism pathways, suggesting that they are involved in these pathways of pharmaceutically important. Thirteen homologous fragments of key genes identified were referred to these pathways. In addition, a total of 6 304 SSRs were identified from the sequence of transcription, distributed in 5 306 unigenes(15.64%). This work not only provides many valuable basal data for gene cloning and molecular biology research, but also lays the foundation for genetic diversity analysis and development of molecular marker in Lepidium apetalum.
Objective: To compare the activity and kinetic parameters of meso-2,3-Butanediol dehydrogenase (BDH) from Enterobacter aerogenes (E. a-BDH) and Bacillus subtilis (B. s-BDH), and analysis the influences of the residue D194 on catalytic properties of BDH. Methods: E. a-BDH and D194G B. s-BDH were expressed in E. coli BL21 (DE3), and purified by HiTrap Q FF anion-exchange and Superdex 75 gel column. MALDI-TOF MS was used to determine the molecular weight. The enzyme activity, coenzyme and substrate specificity, optimum pH, temperature, and kinetic parameters of BDH were investigated by monitoring changes in absorbance of NADH/NAD+ redox reaction. Results: The recombinant E. a-BDH and D194G B. s-BDH are homo-tetramer. Their nucleotide sequences exhibit two different bases (g.27A/T and g.581A/G), and g.581A/G results in an amino acid change (p.D194G). D194G B. s-BDH activity is about 2.3% of E. a-BDH, and lost the ability of oxidation of meso-2, 3-butanediol. Acetoin/NADH is the optimal substrate of BDH, but Km of D194G B. s-BDH is 5.63 times greater than that of E. a-BDH. Conclusion: D194G mutation reduces the BDH activity.
Autophagy is a cellular activity of bulk degradation of cellular components through lysosome. Many autophagy-related genes (ATG), which involved with autophagic process, have been discovered. But many details in this conserved cellular process still remain unknown. The discovery of new molecules involved in the autophagic process will shed insightful light on our knowledge about this cellular metabolic process. A fusion expression recombinant (p62/SQSTM1-luc) combined with both selective autophagic substrate SQSTM1 (Sequestosome 1) and firefly luciferase reporter gene was constructed. SQSTM1-luc was tested as a qualified luciferase-based autophagy-monitor method. This method was optimized by the construction of stable SQSTM1-luc expressing HeLa cells. It has been proved that this is a convenient method that is applicable to screening for autophagy-related genes.
The novel biotransformation method is developed for preparing L-asparagine. Asparagine synthetase gene A (asnA) from Escherichia coli (E. coli) JM109 is amplified and transformed into E. coli BL21 (DE3) using molecular biological methods, and then the ASNA is expressed. After analyzing the ASNA activity, the constructed strain E. coli BL21 (DE3)/pET28a (+)-asnA is applied on conversion from L-aspartic acid to L-asparagine. The substrate and product are detected by PITC column derivatization-high performance liquid. The molecular weight of expressed protein is of about 37kDa by SDS-PAGE, which is consistent with the expected size. The enzyme activity is 1 786.6U/g wet cell. The conversion ratio of L-aspartic acid is 95.8%, the yield of L-asparagine is up to 126.5g/L, and the production rate is 15.81g/(L·h). E. coli BL21 (DE3)/pET28a (+)-asnA engineered strain is constructed successfully. Then it is used to catalyze the conversion of L-aspartic acid to L-asparagine, and ATP is not necessarily in the production process, which results in the lower cost and provides a novel green method for preparing L-asparagine.
In fed-batch culture of Saccharomyces cerevisiae, excessive glucose addition leads to much ethanol accumulation, destroying structure and function of cell and decreasing glucose utilization efficiency, while insufficient glucose addition limits cell growth. To solve this problem, a self-adaptive control strategy based on differential evolution algorithm was proposed. In addition, performances of the proposed strategy, traditional strategy, were tested and compared using computer simulation. As a result, under the proposed control strategy, ethanol concentration could be maintained at the low level of 1.0g/L, while the biomass concentration could reach to the high level of 34.45g/L, which was 243%, 18% and 29% higher than those under intermittent feed, stepped constant feed and PID control strategy, respectively. In conclusion, the proposed self-adaptive control strategy was capable of controlling glucose feed rate at proper level, and thus ensured the rapid growth of yeast when repressing ethanol accumulation.
Endoplasmic reticulum (ER) is the site of protein synthesis, protein folding, maintainance of calcium homeostasis, synthesis of lipids and sterols. Genetic or environmental insults can alter its function generating ER stress. During ERS, protein misfolding and accumulation in the ER lumen initiate unfolded protein response(UPR) through a series of signal transduction pathways that produce various effects, including enhancing the ability of proteins to fold properly, accelerating protein degradation, increasing the probability of cell survival, and strengthening the selfrepair ability of the ER. Either ERS persists or activated excessively, eventually initiates cell apoptosis. Therefore, ER stress and UPR are implicated in the development of various diseases. Recent studies have demonstrated that ER stress and UPR signaling are involved in cancer, inflammatory diseases, metabolic disorders, osteoporosis and neurodegenerative diseases. However, the precise knowledge regarding involvement of ER stress in different disease processes is still debatable. Here the possible role of ER stress in various disorders on the basis of existing literature is discussed.
Using plant as bioreactor to produce various valuable proteins or peptides is the central part of molecular pharming .This method is more and more used because of its advantages such as simple operation, low cost, large scale planting and short period compared with animal and microbe bioreactor. The common optimization methods of vector construction and foreign gene, variety of plant acceptor and the different expression systems of plant bioreactor were reviewed. As the increasing demand of biopharmaceuticals, there is a great perspective of plant bioreactor to be used in molecular pharming in the future.
As a kind of important economic crops, tomato has very-high nutritional value, and is always one of the model organisms to study the growth and maturation of fleshy fruit. In recent years, many tomato fruit ripening mutant were found, which provides important biomaterials for clarifying mechanism of fruit ripening. The factors affecting tomato fruit ripening, fruit ripening related mutants and researches of fruit ripening based on these mutants to provide reference for future researches of mutant and fruit ripening mechanism.
Steroidal alkaloid is one kind of metabolite existed widely in medicinal plants, which is one of natural products with antihypertensive effect, relieving cough and asthma, anticholinergic and other biological activity. At present, the researches on biosynthetic pathway, extraction and purification, identification and biological function of steroidal alkaloid have become a hot point in the natural product field worldwide.The pharmacological effects, [f24]the biosynthesis pathway, involved key enzymes and genes cloning of steroidal alkaloids was summarized according to terpenoids biosynthetic pathway, which will provide a reference for the studies of metabolic pathway, gene expression and regulation and its application of steroidal alkaloid in medical plants.
Pichia pastoris expression system is a new foreign protein eukaryotic one which has been the rapid development of the technology in recent years, widely used in many different fields and many gene engineering products has been successfully expressed. The composition of Pichia pastoris expression system was introduced mainly from the aspects of the expression of common strain, vector and phenotype. It was elaborated in detail that how these effect the process of high cell density fermentation of Pichia pastoris and the result protein expression from the aspects of exogenous gene's own characteristics, the composition of culture medium and the tempreture, pH, dissolved oxygen and feeding strategy. At last, the Pichia pastoris high cell density fermentation was discussed and provides a reference for further research.
Taking Derwent innovation index as the database, chooses bioreactor patents in the pharmaceutical field to analyze time,region, research activity, research field. By using CiteSpaceIII, comparative analysis on bioreactor patents are conducted through Derwent Manual Code Co-occurrence Networks,which reveal to detect technical cooperation relationship,hot technology field,the cutting edge of bioreator.Providing the necessary intelligence supports to enterprise, such critical intelligence supports could help them drawing up technical develop plans, adjusting technical develop direction, improving the overall arrangement of technical develop and maintaining a high technological competitive advantage.
Scientific evidences of genetically modified (GM) crops were investigated based on the Science Citation Index literature from Web of Science data. It shows that scholars pay more attention on safety of transgenic technologies than consumers. In addition, those transgenic plants which have been released for commercial production have been most strictly tested on their biosafety and its rigorous regulatory system has been established. Ninety percent of all the 9333 research papers illustrated no significant difference of safety between transgenic technologies and non- transgenic crops. The conclusions derived from the papers, which hold unsafe view of GM food, were proved to be false because of its wrong research materials or methods.
