Objective: To establish the recombinant L6 myoblasts with exogenous FGFR2IIIc by lentivirus and to distinguish their characters. Methods: FGFR2IIIc gene was obtained by RT-PCR from human placental and was cloned into pENTA-11. Recombinant expression vector pLenti6/V5-DEST-FGFR2-IIIc was obtained by using pENTA-FGFR2IIIc and pLenti6/V5-DEST plasmids with LR recombinase. Then such vector and Viral Packaging Mix vectors were co-transfected into 293FT cells to gain recombinant lentiviral particles. After infected by Lentivirus,stable L6 cell monoclones were selected by blasticidin for 2~4 weeks. The recombinant cell lines with exogenous FGFR2IIIc were identified by RT-PCR,indirect immunofluorescence and Western blot. Co-stimulated by both bFGF and HSPG, cell cycles of recombinant L6 cells were evaluated by flow cytometry. Their myogenic differentiation was observed with or without inhibitors of ERK and p38 signal pathway. Results: The vector pLenti6/V5-DEST-FGFR2-IIIc was reconstructed to gain lentiviral particles to establish recombinant L6 myoblasts with exogenous FGFR2IIIc.The recombinant L6-FGFR2IIIc group show an increase in the percentage of cells in the G1/G0 phase compared to the L6-vector group. Phosphorylated Erk1/2 inhibition with PD98059 leads to some cells myogenesis;And phosphorylated p38 inhibition with SB203580 leads to cells apoptosis. Conclusions: FGFR2IIIc can inhibit L6 myoblasts proliferation and result in the tendency of myogenic differentiation.
Obiective: To study the effects and mechanisms of RMP on cell cycle of human hepatoma cells SMMC-7721.Methods:Construct the shRNA recombinant eukaryotic expression vector pGPU6-Neo -RMP. Establish shRNA RMP stable cell lines;RT-PCR was used to detect the interference efficacy against RMP gene; Flow cytometry was applied to measure the effect of RMP on cell cycle of SMMC-7721,and then the expressions of related genes of cell cycle were detected by RT-PCR.Results: The shRNA recombinant eukaryotic expression vector pGPU6-Neo- RMPi was successfully constructed. The positive monoclones were selected and stably interfered RMP cell line was obtained. Flow cytometry demonstrated that G2/M arrest was discovered in SMMC-7721-RMPi stable cell line. Conclusion: RMP could adjust the cell cycle by adjusting that the G2/M checkpoint relevance gene Cyclin B, CDK1 and P21 expressing.
Objective:Analysis the functional role of p38 kinase in BMP-9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells. Methods:C3H10T1/2 cells were infected by recombinant adenovirus expressing BMP9, then the total protein level and phosphorylated form of p38 kinase were determined by Western blot. After treatment C3H10T1/2 cells with p38 specific inhibitor SB203580 or using RNA interference to silence expression of p38, the early osteogenic marker ALP activity was detected by quantitative and staining assay, later osteogenic marker calcium deposition was determined by Alizarin Red S staining, expression level of Smad6 and Smad7 was analyzed by Real time PCR. Animal assay was carried out to confirm that whether p38 silence can result in inhibition of entopic bone formation induced by BMP9 in vivo. Results: BMP9 did not change total protein level of p38, however, BMP9 increased the phosphorylated form of p38 kinase. P38 specific inhibitor SB203580 dose-dependently decreased ALP activity induced by BMP9 of C3H10T1/2 cells. Gene silence of p38 by RNA interference also led to a reduction of ALP activity. Furthermore, SB203580 markedly inhibited calcium deposition, reduced BMP9-induced expressions of Smad6 and Smad7. Moreover, p38 gene silence was also showed to inhibit entopic bone formation induced by BMP9 in vivo. Conclusion: The p38 kianse may invovlve in BMP9 induced osteoblast commitment of C3H10T1/2 mesenchymal stem cells.
Objective:To explore after indoleamine-2,3-dioxygenase(IDO)gene transfection the influence of the hepatocellular carcinoma cells’ apoptosis and the related cellular immune mechanisms by cell culture and in vivo. Methods: By cell culture and gene transfection technology,T-lymphocytes freshly isolated from healthy people and hepatocellular carcinoma cells were cocultured. The experiments were divided into six groups: T-lymphocytes and HepG2 cells group, T-lymphocytes and pcDNA3.1-HepG2 cells group, T-lymphocytes and pcDNA3.1-IDO-HepG2 cells group,and the three intervention groups which were added to IDO inhibitor 1-MT(1-methyl-tryptophan) on the basis of the above three groups. After two days of combine reaction, the apoptosisrate of HepG2 cell and the cytotoxicity of T-lymphocyte against HepG2 cell were examined by flow cytometer and MTT assay. After five days in mixed culture,the percentage of regulatory T cells(Treg) were analyzed by flow cytometer. Through establishment of the mouse model of human liver cancer cells, the percentage of Treg cells in peripheral blood of mouse was analyzed by flow cytometer. Results: 1.After two days of combine reaction, the apoptosis rate of HepG2 cell and the cytotoxicity of T-lymphocyte against HepG2 cell in IDO-HepG2 group were significantly lower than which in pcDNA3.1-HepG2 and HepG2 groups. They were respectively (1.65 ±0. 14) % and (35.00±2.20)% (p<0.05). With 1-MT groups, the above indexes were significantly higher than before the intervention (p<0.05). 2.After five days in mixed culture,the percentage of Treg cells in IDO-HepG2 group was significant higher and that was(10.53±1.05)%,it was considered statistically significant compared with the control group without 1-MT. In adding 1-MT groups, it decreased significantly (p<0.05). 3.In the mouse model of human liver cancer cells, the percentage of Treg cells in peripheral blood of IDO-HepG2 group increased significantly, that is (15.33 ±1.18)% and compared with the other two groups was statistically significant (p<0.05). Conclusion: 1.Though increasing the percentage of Treg cells in T-lymphocytes, IDO can suppress the apoptosis rate of hepatocellular carcinoma cells and the cytotoxicity of T-lymphocyte. 1-MT can reverse the role of IDO. 2.In vivo test, it can be confirmed that over-expression of IDO can increase the proportion of Treg cells in peripheral blood.
Factor H-binding protein (fHBP) is a novel meningococcal vaccine candidate that elicits serum antibodies that activate classical complement pathway bacteriolysis and also inhibit binding of the complement downregulatory protein, factor H, to the bacterial surface. One limitation of fHBP as a vaccine candidate is antigenic variability, since antibodies to fHBP in the variant 1 (V1) antigenic group do not protect against strains expressing V2 or V3 proteins, and vice versa. It can be defined three domains A,B and C, one epitope expressed by nearly all V1 proteins mapped to the B domain, while epitopes expressed by fHBP V2 or V3 mapped to the C domain. That provided the rationale for engineering chimeric fhbp (which was named fhbp-c2 )molecules containing the full length protein of fHBP V1 and a portion of the C domain protein of fHBP V2 then fhbp-c2 was cloned into the prokaryotic expression vector Pet-30a(+) and expressed in E.coli BL21(DE3).Purified recombinant fHBP-C2 protein stimulated a significant antigen-specific humoral response following two intraperitoneal immunization,while bactericidal assay showed that fHBP-C2 can elicit antibodies capable of inducing bactericidal activity against B N. meningitidis strains.the result indicates that fHBP-C2 is a promising candidate for vaccine development.
Transcription repressors are proteins that bind to specific sites on DNA and prevent transcription of nearby genes through interacting with transcripitonal activators, acting on the basal transcription factor complex or chromatin remodeling. Among the DREB subfamily, there are 6 transcription repressors containing the EAR motif in the A-5 group, functional analysis of some repressor indictaed it could modulate plant response to stress, however their specific functions and mechanisms are reqiured further research. By designing the amiRNA-A5, which could specifically target the mRNAs of these transcription repressors, and transforming the pCAMBIA-pre-amiRNA-A5 expression vector to Arabiopsis, 9 lines of T2 transgenetic homozygotes were got, selected by Hygromycin and PCR. Real-time PCR assays demonstrated that compared to the wild type plants, the relative expression levels of target genes in transgenetic plants were obviously decreased. To analyze the resistance of these transgenetic plants under cold and salt stresses, relative electrolyte leakage (REL) and malondialdehyde (MDA) content were measured. Both of these two indexes were increased less in the transgenetic plants than in the wilde type plants under stress conditions, suggesting the increased resistance of the transgenetic plants to cold and salt stress. The results indicated these transcription repressors might negatively function under cold and salt stress, provding clues for further functional mechanism study of the transciption repressors of the A-5 group among the DREB subfamily.
A 5' flanking sequence of PtSEP2 which is a SEPALLATA2 -like gene involved in floral organ development was amplified by PCR from the genomic DNA of Populus tomentosa. The length of fragment is approximately 2.3kb. The results derived from PlantCARE analysis showed that the sequence contains conserved cis-acting elements of promoter, coupled with a variety of light-responsive elements. Therefore, it was speculated preliminarily to be the promoter of PtSEP2 gene. To investigate function of this promoter, it was fused to GUS reporter gene, generating a plant expression vector pPtSEP2 promoter∷GUS, named PtSEP2p∷GUS . With roots, stems, leaves and flower buds of Nicotiana tobaccum for the receptor, the results of transient expression that Agrobacterium-mediated that showed the PtSEP2 promoter was competent to drive specially GUS reporter gene expression in anther, yet its’ activity was weaker than that of cauliflower mosaic virus (CaMV) 35S promoter, which constitutively expressed in planta. Consequently, provides a possible genetic modification tool for flowering regulation in poplar and other plants.
The vaccinia virus/T7 RNA polymerase transient expression system relying on the bacteriophage T7 RNA polymerase with several key advantages was widely used for protein expression. VP6-encoding cDNA of rotavirus strain TB-Chen was cloned and constructed by inserting into prokaryotic expression plasmid pETL between bacteriophage T7 promoter and terminator regions. When eukaryotic cells were transfected with the recombinant plasmid containing the VP6 gene and infected with recombinant vaccinia virus vTF7-3 which provides bacteriophage T7 RNA polymerase, the VP6 gene was expressed. The findings showed that cytopathic effects by vaccinia virus infection were fast and severe leading to low level of protein expression. If cytopathic effects of the vaccinia virus can be reduced, higher level of the protein might be obtained.The findings provided a helpful means to further study the VP6 function and utilize the vaccinia virus/T7 RNA polymerase transient expression system.
ESAT-6 gene was amplified by PCR from the genome of Mycobacterium tuberculosis H37Rv, and then cloned into pET21a(+) plasmid. The recombinant plasmid that was successfully constructed was transformed into E.coli BL21(DE3). After induced with IPTG, the expressed recombinant protein was confirmed by SDS-PAGE and Western blot. The vector yielded satisfactory levels of recombinant ESAT-6 protein expressed as a soluble protein in E. coli. After ultrasonication, the recombinant ESAT-6 protein was firstly purified by a column packed with Ni-NTA Resin and then a column packed with DEAE-SepharoseTM Fast Flow matrix. The purity of the purified protein was about 95%. The purified ESAT-6 protein was incubated with RAW264.7 cells, and the result got by Immunofluorescence showed that ESAT-6 could directly bind to the macrophage membrane.
The promoter is an important cis-element for regulation of gene expression,the strength of the promoter function is very important for the expression of target genes. In order to find a better promoter for transcriptional initiation, the experiment compared different promoters in the expression of foreign proteins in Pichia pastoris, using the green fluorescent protein (GFP) as a reporter gene.The promoter of glycerol-3-phosphate dehydrogenase gene(Scgpd) which is the key enzyme in Glycerol synthesis from Saccharomyces cerevisiae was cloned by PCR, and introduced it into pPIC9K and pGAPZB to construct pPIC9K-gfp and pPIC9K-PG,pPGAPZB-gfp and pGPDZB-gfp simultaneously. The recombinant plasmids were transformed into Pichia pastoris GS115 and Pichia pastoris X33 by electroporation. In the medium containing glucose or NaCl with different concentrations for culturing the recombinant strains, GFP was detected by fluorescent microscopy. By the expression of GFP,the fuction of pScgpd and methanol oxidase promoter pAOX1, 3-GPDH promoter pGAP was compaired. Fluorescence microscopy showed pScgpd induced by the performance of hypertonic conditions. The gene gfp was functionally expressed under the control of the promoters in recombinant Pichia pastoris. Furthermore, the expression of the gene gfp at different level was conducted by the different concentrations of glucose or NaCl within a certain range for the recombinant strains. However, compared with the promoter pGAP and pAOX1 corresponding to the host P.pastoris GS115 and P.pastoris X33, the expression level under the control of the promoter pScgpd is still weak.
The culture medium and fermentation conditions of erythritol production by Moniliella acetoabutans was optimized by response surface methodology. According to the results of mono-factors experiments, a Plackett-Burman design was used to investigate and evaluate the influence of related factors, three factors are statistically significant: glucose concentration, initial pH value and temperature. Box-Behnken was employed to design. The quadratic regression analysis was conducted on the optimized results by using SAS software, it was found that the optimum conditions for erythritol production were glucose26%, yeast extract 0.4%, KH2PO4 0.2g/L,MnSO4·4H2O 0.05g/L,CuSO4·5H2O 0.04g/L, initial pH value 4.2, temperature 31℃, broth content 20%, shaking speed 150r/min, inoculum 4%. Under this condition, the concentration of erythritol reached to 75.63g/L in the shake-flask experiments after 90h fermentation, increased by 10.8% than before, the yield was 36.12%, increased by 9.4% than before.
Using triose phosphate isomerase deficient (tpiΔ) mutant of Saccharomyces ellipsoideus as the control, the regulation of osmotic press on the balance between glycerol production and volatile acid formation was first studied. The results demonstrated that there existed the control of osmotolerance on the balance between dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) in wild S. ellipsoideus and the suitable high osmotolerance can transport metabolic flux from GA3P to DHAP to produce much glycerol. However, the control of osmotolerance didn't exist in tpiΔ mutant of S. ellipsoideus. In comparison with wild S. ellipsoideus, the tpiΔ mutant greatly reduced the yield of glycerol production and much created glycolytic metabolites because of GA3P accumulation at the same conditions. Therefore, the conclusion was made that, at the suitable high osmotolerance, DHAP accumulation of wild S. ellipsoideus would be helpful to regulate the ratio of glycerol to volatile acid.
Saccharopolyspora spinosa CB11was used as the recipient strain . Optimal conditions including growth stage of the strain, electroshock voltage,DNA concentration were investigated for the electrotransformation of CB11 with Escherichia coli-Streptomyces shuttle vector pSET152.It was showed that the highest electroporation efficiency was yielded under the cluture age of 48h, electric field strength of 12kV/cm and minimum DNA concentration needed 0.1μg . Plasmid stability experiment and PCR of acc(3)IV gene showed that pSET152 was successfully electroporated into and could stably exist in the Saccharopolyspora spinosa CB11. This protocol would be useful for genetic studies of S. spinosa.
Using (R, S)-2-carboxyethyl-3-cyano-5-methyl-hexanoic acid ethylester (rac-CCMAE) as sole carbon source, Pseudomonas sp. CGMCC No. 4184 was isolated which can enantioselectively hydrolyze (R)-CCMAE to (3R)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (3R-CCMA) with eep of over 90%. The optimal temperature and pH for the production of hydrolase by Pseudomonas CGMCC No. 4184 were 30℃ and 7.4. Single-factor experiment, Plackett-Burman design and response surface methodology were applied subsequently to optimize the fermentation medium. After optimization, the enzyme activity increased from 15.92U/L to 33.43 U/L (about 2.1 fold). By using resting cell catalysis system, (S)-CCMAE were successfully prepared with the conversion of 56.3% and ees of 98.3% after 48 h under the condition of 5g/L CCMAE, 8g/L (cell dry weight) resting cells, 20ml 0.1mol/L phosphate buffer (pH 7.4), 30℃ and 200r/min.
Objective: To observe the induction of differentiation and therapeutic effect of insulin secreting cells on rat diabetes. Methods: The bone marrow stem cells were extracted form rats and induced into insulin secreting cells with nicotinamide and exendin-4. 24 Wistar rats were randomized into normal, diabetes, and induction group. The diabetes model were established for the diabetes group and induction group. The induced cells were intraperitoneally injected into rats of the induction group. The weight, blood glucose ( fasting and OGTT 120min ), and fasting serum insulin were measured. At the end of the experiment, liver, spleen, pancreas were removed, and the double positive(insulin and BrdU) cells were calculated by immunohistochemistry. Results: At the end of administration, the fasting serum insulin of induction group was significant higher than the diabetes group (P<0.05), and the fasting and OGTT 120min blood glucose was lower than the diabetes group(P both<0.05). The ratio of insulin positive cells in the induction group was higher than diabetes group, and some insulin and BrdU duble-positive cells were found in pancreas, liver, and spleen of induction group. Conclusion: Nicotinamide and exendin-4 can induce bone marrow stem cells into insulin secreting cells, and these cells can increase the serum insulin, reduce the serum glucose, then improve the rat diabetes.
Enhanced green fluorescent protein (EGFP) gene sequence with 5' end His tag was amplified by PCR. The recombinant baculovirus encoding EGFP was constructed and transfected into sf9 cells by Cellfectine reagent. The resultant fluorescence was observed by fluorescent microscope, and the expressed EGFP protein was purified by Ni column affinity chromatography and exploited as antigen to analyze specific humour immune response. The open reading frame (ORF) of EGFP with 5' end His tag was cloned successfully. The expression of EGFP from recombinant baculovirus was confirmed by fluorescent microscope, and the purified EGFP reached 90% purity and had the ability to bind specific antibody. The high efficient expression, simple protocol available for purification and intact biological activity are the advantages to prepare EGFP from baculovirus expression system.
A phage display peptide library was screened by targeting recombinant p24 antigen. After three rounds of panning, eleven phage clones were chosen to test the binding affinity by ELISA, and ten of them were positive. DNA sequencing showed that nine of the positive clones display a unique sequence FTTRANA which has high affinity with HIV-1 p24 antigen. To improve the condition of phage ELISA, phage number and blocking buffer have been optimized, phage number ranged from 8.13×1011 to 1.02×1014 pfu/well and 0.5%BSA was highly recommended. Therefore, in this study, peptides combined with p24 antigen were obtained which might be useful in p24 antigen assay.
Recombinant protein EH originates from hirudin by linking three amino acids to its N-terminal. The three amino acids are recognized and cut by coagulate factor Xa (FXa) and XIa (FXIa), that will release the antithrombin activity of hirudin.The cleavage efficiency of FXa to EH was compared under different conditions, incuding emzymolytic time and temperature, the molar ratio of FXa and EH, and reaction solution. Results showed that the antithrombin activity of EH increased proportionally, under certain conditions, as the reaction time and temperature increased, Moreover, the FXa cleavage activity is enhanced when the molar ratio of FXa and EH was increased. It seems that saline is more suitable reaction solutions for FXa to cut EH. So the optimal cleavage conditions for EH with FXa in vitro is that EH was reacted with FXa at 37℃ for 6 hours in saline solution with the molar ratio (1 ∶180) of FXa and EH. A practical and reproducible method for the activity determination and quality control of EH in vitro was developed.
In recent years, various kinds of HIV entry inhibitors have been invented with the discovery of the HIV entry process. They are divided into three main classes: attachment inhibitors, co-receptor binding inhibitors, and fusion inhibitors. The mechanism of action and structure of several important inhibitors are discussed. Many inhibitors are in advanced clinical trials. Especially, the fusion inhibitor T20 has been approved for the treatment of the HIV infected patients with other antiretroviral therapys,by FDA,in 2003 and the CCR5 inhibitor maraviroc has been approved by FDA, in 2007.
In order to meet consumers' right to know for genetically modified(GM) food,it is very important to establish accurate, rapid and efficient GM food detection technology. The quality and quantity of the DNA is considered to be a prerequisite factor for the GM food detection.Several DNA extraction methods used at home and abroad in the recent years are reviewed, such as hexadecyl trimethyl ammonium bromide (CTAB) methods, dodecyl sulfate, sodium salt(SDS) methods,polyvinylpyrrolidone( PVP )methods,medium purify methods,chelating resin methods etc. The advantages, disadvantages, application range of the above methods are comprehensively summarized, along with analyzes the problems should be paid attention to and the GM food DNA extraction developing direction, which will offer operative information for rudimenting workers in DNA extraction.
Diatoms are microscopic, single-celled algae that possess rigid cell walls (frustules) composed of amorphous silica. The intact diatom frustules possess intricate nanoscale features. Depending on the species of diatom and the growth conditions, the frustules can display different morphologies. The almost 200 000 different diatom species feature unique frustule architectures that are instructive for construction of photonic structures, chemo/biosensing and new nano-devices. The researches of diatom molecular biology and frustule formation are instructive for development of biomimetic synthesis of silica-based materials, chemical transformations and templating techniques. It is possible to design and produce specific frustules that have a wide rang of applications in nanotechnology.
Lactobacillus reuteri is reported to inhabit the gastrointestinal tract of almost all kinds of vertebrates and mammals by establishing a symbiotic relationship with other bacteria. The adhesion and colonization capability of Lactobacillus reuteri to the mucosa of gastrointestinal tract was introduced here. Reuterin-a wide-spectrum antibacterial substance which is produced by Lactobacillus reuteri and probable acting mechanism of Lactobacillus reuteri was described. The health effects of Lactobacillus reuteri to human and animals were especially emphasized and the industrial development of Lactobacillus reuteri probiotics in future was also discussed.
Veterinary vaccine is veterinary medical product, so it needs the same or similar regulation and control standards as medical product. And animal vaccine must meet the complex requirements, ensuring the quality, safety and immune efficacy. In this field, biological products have more problems, because biological products are basic with natural existing organisms, so they are easier to be imitated and not protected by the patent law. In the animal medicine field, the animal vaccine regulation and control system of the European Union and the United States develops early, and by now it has almost been integrity. It can offer valuable reference informations for other countries and regions to establish good animal vaccine regulations and control standards. In addition, that unifying all countries and areas' veterinary vaccine regulation and control standard is a way to accelerate making free, rapid global veterinary vaccine international trade.
