Objectiive:The present study was designed to explore whether overexpression of human wild α-synuclein in rat brain caused selective dopaminergic neuron loss in substantia nigra and aimed to find out a new method to make a rat model of Parkinson’s disease (PD). Methods:The human wild α-synuclein gene was induced into the rat brain by AdenoAssociated Virus (AAV) vector. The overexpression of α-synuclein was detected by realtime PCR. The behavior of rats were recorded every 4 weeks after the viral particle injection. TH immunohistochemistry were performed at 4, 8, 12 and 16 weeks post-injection as well as the dopamine (DA), 3,4-dihydroxypheny lacetic acid (DOPAC) of striatum were determined by high performance liquid chromatography coupled with electrochemical detection. Results:Realtime PCR results revealed a significant overexpression of α-synuclein in the injected hemisphere. By 8 weeks post injection, a significant loss of the dopaminergic neurons was observed. 34% of the dopaminergic neurons were lost after 12 weeks, and about 60% cells loss after 16 weeks. The DA and DOPAC levels in the striatum decreased about 15% 12 weeks after injecting viral particle carried α-synuclein gene and 30% decreased after 16 weeks. The AAV-α-synuclein-treated rats developed a type of motor impairment, i.e., head position bias, compatible with this magnitude of nigrostriatal damage. Conclusion:All the results showed that overexpression of human wild α-synuclein caused selective dopaminergic neuron loss and mimic a symptom of human PD in rats. This may be a new methed to make rat PD model which can offer new opportunities for the study of pathogenetic mechanismsand exploration of new therapeutic targets of particular relevance to human PD.
Objective:To construct pGL3 Basic eukaryotic expression vector containing tissues specific promoter of FLT-1 and explore the activity of this promoter in HUVEC cells. Methods: The FLT-1 gene promoter was amplified by polymerase chain reaction and cloned into pGL3 Basic vector to construct pGL3 Basic eukaryotic expression vector containing FLT-1 gene promoter(pGL3-FLT-Basic-luc)．The purified pGL3-FLT-Basic-luc was transiently transfected into HUVEC cell and HepG2、NIH3T3、HEK293 cell using liposome transfection reagent, and the activity of luciferase was determined with Dual-Luciferase Reporter Assay System 48h later．Results: DNA sequencing and digestion confirmed that the recombinant of plamid pGL3-FLT-Basic-luc contained FLT-1 promoter sequence.The activity of luciferase in HUVEC was much higher than in HEK293 after transfection of pGL3-Basic-luc, and little activity of luciferase was detected in other two cells．Conclusion: pGL3 Basic eukaryotic expression vector containing tissues specific promoter of FLT-1 was successtully constructed，which might be a potential therapeutic reagent for endothelium-targeted gene therapies for vascular disease.
P300/CBP-associated factor (PCAF), an important member of histone acetyltransferase family (HATs) within eukaryotic cells, is capable of inducing the acetylation of histone, promoting the transcription of specific genes and involving in many biological effects. In the present study, fulllength cDNA of PCAF was inserted into plasmid pGEX-5x-1, then the soluble protein GST-PCAF was expressed in E.coli BL21(DE3) after the optimization of inducing conditions. The recombinant protein was further purified with affinity chromatography and tested the activity by in vitro acetylation assays. High efficient PCAF protein produced by this method could serve for the study on the role of PCAF in gene regulation and the interaction between PCAF and other proteins
To analyse the bioactivity of α-2,6 sialyltransferase protein, the total RNA was extracted from HepG2 cell and the α-2,6 sialyltransferase gene included an open reading frame coding for 406 amino acids was amplified using RT-PCR. Then the amplified products were inserted into pMD-18T vector. After sequenced, the target DNA fragment was cloned into pET
To study the activity of a specific bFGF-binding phage selected from a phage display heptapeptide library. ELISA was applied to assess the bFGF-binding curve of the positive phage clone obtained from 3 rounds screening. The competitive binding activity of the bFGF-binding phage was examined by competitive inhibition assay. The effect of the bFGF-binding phage on the proliferation of Balb/c 3T3 cells induced by bFGF was investigated by MTT method. The positive phage selected from the phage library specifically band to bFGF in a dose-dependent manner, and its binding with the immobilized bFGF was competitively inhibited by free bFGF. The isolated phage could also inhibit the proliferation of Balb/c 3T3 cells induced by bFGF. The bFGF-binding phage could inhibit bFGF activity, which would offer the basis for further developing the anti-tumor short peptide targeting bFGF.
The salt damage to the plants is mainly caused by Na+. The catalytic transport of Na+/H+antiporter protein causes Na+ to come out of the Na+ compartmentalization of the vacuole membrane and the plasma membrane of the cell. The halophyte plants have been found to be resistant to salt stress caused by Na+. The conservative transport protein gene sequence of halophyte, Plantago maritima was carried out first time at Xinjiang by using RT-PCR and RACE technology and cloned into the Na+/H+ antiporter transport protein gene (2464 bp full-length cDNA) named as PmNHX1 (GenBank accession number: EU233808).It was found that the head of the gene consist the coding of the 1662 bp which encoded 553 amino acids. It had the molecular weight of 61.16 KDa and isoelectric point was 7.22. The data analysis showed that the protein mainly located in vacuole membrane was from 12 conservative sequence of the transmembrane domain of which the TM3 transmembrane domain (LFFIYLLPPI - putative amiloride binding domain) was found to be responsible for playing a competitive role. The amino acid homology of PmNHX1 and other plants antiporter protein was 64% - 80%. Due to its unique ability to find out physical and chemical properties and forecast the function of the gene, the use of bioinformatics methods laid the foundation for study related to identification of the salt-tolerant gene function of PmNHX1.
Abstract An α-Cyclodextrin glucanotransferase(CGTase) from Bacillus sp.602-1 was immobilized on DEAE cellulose by covalently binding with glutaraldehyde. Key parameters for immobilization, such as concentration of glutaraldehyde, amount of enzyme and immobilization time, were optimized respectively. Properties of the immobilized CGTase were further investigated. No difference was observed on the optimal temperature between free enzyme and immobilized one, but the optimal pH of the latter shift from 6 to 5. pH and thermal stability of the immobilized CGTase increased to some extent. The yield of cyclodextrin reached 32% after conversion by the immobilized CGTase on starch at 40℃ for 3h, 150r/min. The immobilized CGTase could be used for 4 cycles continously. After being stored for 18 days at 4℃ in 5mmol/L CaCl2 solution, residual activity of the immobilized CGTase remained 80%.This work will be helpful for the industrial application of the α-cyclodextrin.
1,3-propanediol (1,3-PD) is an important material for chemical industry, therefore, there is much interest in the production of 1,3-PD. The genes dhaF and dhaG encoding glycerol dehydratase reactivase were amplified by using the genomic DNA of Citrobacter freundii. Two segment genes about 1.8Kb and 0.4Kb were obtained and inserted into pMD-18T. There had not more than 86% similarity between the cloning genes and other corresponding genes; then dhaF and dhaG were inserted into pSE380 and co-expressed in E.coli. The interesting recombinant reactivase was about 30 per in the whole crude protein and was purified homogeneous. α and β subunits of reactivase had apparent molecular masses about 63 and 12 kDa by SDS-PAGE, nondenaturing PAGE analysis the molecular mass was about 150kDa. Therefore, its subunit structure was most likely α2β2. In the presence of free adenosylcobalamin, ATP, and Mg2+, the factor reactivated glycerol dehydratase from C. freundii, which had been inactivated. This research is help to make clear the mechanism of reactivase from C. freundii and improve the manufacture of 1，3-PD by the biological pathway.
The biosensor based on the principle of surface plasmon resonance (SPR), is a surface sensitive optical device for monitoring biomolecular interactions at the sensor surface in real time without any labeling. It is being used in a wide variety of areas such as proteomics, drug discovery, clinical diagnosis, food analysis, environmental monitoring and so on. The sensor chip is the core of the Biacore instrument. Since sensor chips can now only be purchased from Biacore AB (Uppsala, Sweden), which makes them expensive, many instruments are either used at a low rate or sleeping. In this paper, Simple and cost-effective methods for the regeneration of five kinds of sensor chips for the Biacore instrument, which include the J1, C1, CM5, SA and NTA sensor chip, were described. And several applications of these regenerated sensor chips were listed. Through the improvement and optimization of these methods in the last four years, the regenerated sensor chip can achieve the same quality as the Biacore sensor chip. The wide use of these regeneration methods will be highly beneficial to the application and development of SPR technology in a wider variety of areas.
Mixed anhydride(MA) was used to conjugate ractopamine (RAC) to bovine serum albumin(BSA) and obtained artificial antigen BSA-RAC identified by UV and SDS-PAGE. Balb/c mice were immunized with BSA-RAC and hybridoma lines that secrete RAC monoclonal antibody(mAb) were generated with cell fusion. A ciELISA kit for detection of RAC (RAC-Kit) was developed with RAC mAb and its traits were tested. The results indicated that BSA-RAC was synthesized successfully and its conjugation ratio of RAC to BSA was about 24.5∶1. Three hybridoma lines were filtered and the best one was 4D8, its affinity constant (Ka) was 1.65×1010L/mol. The detection limit of RAC-Kit was 0.5ng/ml and its detection range was 0.5 to 151ng/ml. The recoveries of RAC spiked in feed were 87.2% and in swine urine were 89.4%. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. It had 22.3% cross-reactivity (CR%) to dobutamine and little or no CR to other compounds. The validity of RAC-Kit in 4℃ was above 180d.
Mutants of recombinant hIL-2 (rhIL-2), generated by using site-directed mutagenesis strategy, can increase anti-tumor activity and decrease toxicity. We have used site-directed mutagenesis of hIL-2 to generate a mutant of hIL-2(MhIL-2) in which Asn88 was substituted by Arg88. To reduce the undesirable formation of inclusion body and maximize the yield of soluble MhIL-2 fusion protein, we adapted a protocol for the expression of soluble MhIL-2 fusion protein. Our results have indicated that soluble form of the MhIL-2 fusion protein is expressed from E. coli.. Moreover, it also has facilitated purification of the MhIL-2. SDS-PAGE analysis revealed that the MhIL-2 protein was efficiently purified to 95% purity by the combination of nickel ion Chelating column chromatography, Desalting column chromatography, thrombin cleavage and Superdex 75 gel filtration column chromatography. Proliferation assay of T lymphocyte of purified MhIL-2 showed that the biological activity of MhIL-2 was higher than that of standard hIL-2 in vitro. This work not only describes an efficient preparation strategy of MhIL-2, but also introduces a highly active MhIL-2 that may have important clinical applicability.
A turbid solution was formed through the reaction between γ-PGA and cetylpyridinium chloride (CPC) and the turibidity was measured by using ultraviolet-visible spectroscopy at 680nm The linearity between the concentration of γ-PGA and its absorbance ,the stability ,repeatability and recovery of the method were studied. Through the reaction of γ-PGA with CPC ,a homogeneous turbid solution was formed .The turbid solution was stable in 3h under proper pH value and ion strength ,the absorbance of the solution at 680nm had a good linear relationship with the concentration of γ-PGA in the range 12.5-50ug/ml (R2=0.9939).The recovery was within the range of 86%-99.75% (n=5)The relative standard division of the method for determining γ-PGA at the concentrations of 5 、10和 40ug/ml were 0.14%，0.23%和0.025% repeatability. The turbidmetric method has advantages of convenience ,simplicity and good repeatability and can be used to control the quality of γ-PGA and its products.
The aim of the study was to construct the recombinant vector with double report gene of EGFP-LacZ which was expressed in eukaryotic cells. LacZ gene was amplified by PCR with pLenti6/V5-LacZ plasmid as template. The PCR products of LacZ gene were digested by restriction enzymes of EcoRⅠ and BamHⅠ, and linkaged with eukaryotic expression vector pEGFP-C1. The recombinant plasmid, named pEGFP-C1-LacZ, was identified by PCR and restriction analysis. pEGFP-C1-LacZ was thansfected into the eukaryotic cells of 293 FT and chicken embryo fibroblast (CEF) by lipofectin. The expression of EGFP-LacZ double report gene was detected by observing the green fluorescence and staining for beta -galactosidase activity. Green fluorescence was detected by fluorescence microscope in transfected cells. Moreover, the positive cell was observed by histochemistry of beta-galactosidase activity. The data indicated that pEGFP-C1-LacZ containing EGFP-LacZ double report gene has been constructed and expressed in eukaryotic cells successfully. The results would contribute to overcome the limitations and uncertainty caused by using of single report gene as molecular marker.
To construct a secretary-expression vector of antimicrobial peptide Bactenecin 7 (Bac7), transform it into Lactococcus lactis MG1363, and identify the expression product and its bioactivity. The DNA sequence of Bac7 and its regulation elements was synthesize with overlap-extension PCR. Then the interest gene was cloned into the shuttle-vector pMG36e after it was digested by Sac I and Hind III, the recombination vector was transformed into Lactococcus lactis MG1363 with electrophoration. RT-PCR and Western blot assays were applied to investigate the expressions of the Bac7 gene, and the bioactivity of the expression products Bac7 in culture supernatant was tested with plate-diffusion method. According to the DNA sequencing and double enzyme digestion results, the recombinant vector was successfully constructed and transformed into Lactococcus lactis MG1363, Lactococcus lactis MG1363 carried the recombination vector could secrete bioactive antimicrobial peptide Bac7. All these results indicate the recombination lactic acid bacteria can expression and secrete bioactive Bac7 efficiently, which lay a foundation for further study of oral administration of a Bac7-secreting lactic acid bacteria to treat intestinal bacteria infection.
Hair follicle stem cells located bulge region of hair follicle, had characteristics of all adult stem cells, including slow-cycling, undifferentiated. They also had the abilities of self-renew and proliferation in vitro. CD34、K15、K19 and Nestin might be the makers of the hair follicle stem cells. They could be induced to differentiate to neurons, glial cells, keratinocytes, smooth muscle cells ,melanocytes In vitro and neurons, melanocytes in vivo. There were many signals in regulating the hair follicle stem cells involved Wnt signal、BMP signal and NFATc1etc.
Abstract：Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets, which could amplify templete RNA in 2h under isothermal condition at about 42°C and without any special equipment. NASBA is now widely applicated in diagnosis of many pathogenic microorganism. This article is mainly about principles and applications of NASBA in viral diagnosis.
Cellulase system contains a series of complex components. There are still some problems remained unclear in cellulase and its mechanism of hydrolyzing lignocellulosic materials and its hydrolysis kinetics, so profound study is needed. The rapid development of many kinds of new interdiscipline such as biochemistry, molecular biology and gene engineering, has further clarified the structure and function of cellulase, and the relationshi Pof its gene expression and regulation, and furthermore resulted in derivative study methods about cellulase in more aspects. Cellulase system components according to synergistic catalytic mode and the sequence of the homology amino acids similarity, summarizes traditional detection methods of enzyme components, and emphasizes on research progress of various biosensors applied in detection of cellulase activity and gene expression was introduced.
Various cancers have seriously threatened human’s health. Screening newer and more effective anticancer agents from natural sources to cure these diseases is the focus in research. As novel sources of potential medicine, a number of metabolites isolated from endophytic fungi have been proved to have anticancer bioactivity. Usually, these endophytic fungi have special biochemical pathway and they can accumulate anticancer agents in cultures such as taxane, alkaloids, cytochalasins, podophyllotoxin, brefeldin A and so forth. The research advance on anticancer agents purified from endophytic fungi is expatiated systemically. In addition, the strategy of screening anticancer agents as well as the prospect on this area is introduced briefly.
Lipases have catalytic active in both aqueous phase and the non-aqueous phase and have a wide range of application in various industrial areas. However, the high cost of lipase production has restricted its extensive use in industry. Solid state fermentation possesses many advantages, such as low requirement for devices, low energy consumption, low production cost, little pollution to environment and easily being popularized, which have made it an important means in microbial production of lipases. Owing to the rapidly increased energy cost and the people's awareness of environmental protection, the solid state fermentation technique, which was regarded as low-tech in the past, has regained attention and developed rapidly since the 1990s. This paper reviews the production of lipase by SSF technique．Mainly contents describe its characteristics, including physical and chemical factors and bioreactors.
Aspergillus oryzae is an important microorganism, which was widely used in the fields of food, brewing, pharmacy and fermentation industry etc and was termed as a generally regarded as safe(GRAS) organism by the United States Food and Drug Administration (FDA).The strategies for promoting the expression of homologous and heterologous proteins in Aspergillus oryzae were reviewed. Which included the usage of strong promoters, multicopies of coding genes, optimization of culture medium and overexpression of the hemoglobin domains (HBD) etc. Heterologous proteins were usually exhibited low expression in Aspergillus oryzae due to the degradation by the proteinases from host. Therefore, development of proteinase auxotrophic stains of Aspergillus oryzae as host is neccessary. In addition, fusion of the heterologous protein with a highly secreted protein of Aspergillus oryzae was an alternative way to prompt the expression of heterologous protein in Aspergillus oryzae.
Hydrogen is a kind of ideal clean energy sources．With low energy consumption，environmental protection and other advantages，biological hydrogen production technology become the hotspot of current study home and abroad．Considering from energy source point of view and environment point of view，it is very important to develo Ptechnology of hydrogen production from biomass．It attempts to analyzed the research of biohydrogen in foreign countries from 2000 to 2008 and drew its knowledge mapping. The knowledge mapping displays that there are two research groups on biohydrogen．The first research grou Pconcentrate on photoproduction of hydrogen from water,including algae and cyanobacteria；The second grou Pfocus on anaerobic fermentation of hydrogen, including photofermentation and dark fermentation．The ma Pshows the researchers of anaerobic fermentation of hdyrogen is the major category in biohydrogen.
