The gene of human Islet neogenesis associated protein (INGAP）was amplified with RT-PCR and cloned into prokaryotic expression vector pET22b(+). INGAP was expressed in the E.coli BL21(DE3) and purified by affinity chromatography and gel filtration chromatography. The inclusion bodies of the expressed protein were extracted and dissolved in 8mol/L. The heparin Agrose affinity chromatography was used to separated the desired protein, and the further purified protein was obtained by the Superdex75 the gel filtration chromatography. The purified INGAP protein immune the rabbits by injection,and the polyclonal antibody against INGAP protein was prepared. The immunological activity of expressed and purified LexA protein was detected by ELISA , and Western blot. The result showed that the INGAP was accounted for about 40 % of the total bacteria protein. The final purity of the INGAP was 98.81%, which was determined by the HPLC. The expressed and purified LexA protein had satisfactory immunological activity, which was confirmed by immunodiffusion and ELISA
Many bioactive peptides from neural and endocrine tissue are amidated at C-terminals, which is essential for their activities. The α-amide comes from post-translational modification that is catalyzed by α-AE (α-amidating enzyme) or PAM (pepdilylglycine α-amidating monooxygenase). In this paper, the gene encoding α-AE was amplified with PCR and cloned into the plasmid pET-30a. After the recombinant plasmid pET-A was transformed into E.coli BL21, the α-AE was expressed and purified by the Ni2+ affinity chromatography, which has the ability catalyzing Dns-Tyr-Val-Gly to Dns-Tyr-Val-NH2. It identified that the recombinant protein producing by E.coli BL21 is α-AE, which will benefit for studies of amidation at the C-terminals of peptides.
To construct eukaryotic expression vectors that utilize human CD46 promoter to drive expression of genes, hCD46 promoter was amplified by PCR using genomic DNA from HeLa cells as template. The PCR product of 1.5 kb was subcloned into pMD18-T vector and submitted to sequence analysis. Nucleotide sequence alignment showed that the self-cloned hCD46 promoter was 99.9% homologous with a DNA fragment from the 5’ end of hCD46 gene published in 2006, differing only in 2 nucleotides. The hCD46 promoter was used to replace the CMV early promoter in pcDNA3EGFP; and the rabbit β-globulin gene intron 2 (RGI) was inserted between hCD46 promoter and EGFP gene in order to enhance the expression level of the gene of interest. The recombinant expression vectors, pCDPEGFP and pCDPEGFP-RGI, were transfected into CHO and SP2/0 cells, respectively. Detection by FACS revealed that the expression level of EGFP in transfected CHO cells was higher than that in transfected SP2/0 cells, similarly to the expression property of hCD46 in vivo in human. RGI could enhance the expression level of EGFP in each cell line without altering the cell line-specific expression characterization. These data indicate that the eukaryotic expression vectors containing human CD46 promoter could be suitable for the development of transgenic mice that mimic the expression properties of hCD46.
Object: To study the proliferation of hCTGF on cells and its biological function on bone injury healing. Methods: The fibroblast with potential differentiation transfected by eukaryotic gene delivery system was transferred into the experiment animal with bone fracture. The data were attained by molecular biology and clinical orthopedic technique detection analysis. Results: The results demonstrated an obvious proliferation of hCTGF on cells, suggesting that hCTGF have the biological activity of repairing bone injury via gene therapy approach. The observation provides a new activity factor and new treatment approach for bone injury in clinics.
Objective: Aim: To prepare chitosan nanoparticlescarrying gene and study its characteristics in vitro and vivo. Methods: 12-ACSs was dissolved in 0.05 M sodium acetate buffer to form a solution of 1 mg/mL and a DNA(plasmid-encoding antisense ECE ) solution of 0.1 mg/mL was dissolved in 25mM Na2SO4. The charge ratio of components is selected as 2/1 for 12-ACSs /DNA complex. The complex was prepared by mixing 12-ACSs solution with DNA solution prior to observation by using transmission electron microscopy.Using Electrophoretic Retardation of DNA Migration detection, DNA precipitation, Binding Equilibration and DNase Resistivity assay, the formation of 12-ACSs /DNA complex was determined and its stability was simultaneously evaluated. MTT Cell Proliferation Assays was performed on investigation of the cytotoxicity of 12-ACSs /DNA nanoparticles in bronchial epithelial cells (16HBE). The transfection efficiency of 12-ACSs /DNA nanoparticles in vitro and in vivo was investigated in 16HBE cells and Balb/c mice. Results: 12-ACSs /DNA nanoparticles (100~150nm) were observed by transmission electron microscopy.12-ACSs can protect the plasmid encoding antisence-ECE from degradation by DNase I.12-ACSs can transfer plasmid-encoding antisense ECE into 16HBE cells and into the airway epithelium in mice. As shown by fluorescent microscopy, green fluorescent protein reporter can be observed in the transfected cells as well as in the airway epithelium of the treated mice. And it showed a lower cell cytotocixity in cultured 16HBE cells and in mice treated with12-ACSs /DNA nanoparticles. Conclusion: In summery,the chitosan can be used as an effective protectant for DNA as well as an enhancer for in vitro gene transfection. Key words: asthma, chitosan, nanoparticle, Endothelin converting enzyme, gene therapy
Tobacco mosaic virus (TMV) RNA and Cucumber mosaic virus (CMV) were extracted and sequenced, respectively. The full-length sequence clones of their coat proteins were therefore obtained using reverse transcription method; the target coding region sequence obtained using forward and reverse primers, was later subcloned into pUCCRNAi, then transferred into pCAMBIA2300-35S-OCS expression vector. Utilizing Agrobacterium tumefaciens LBA4404 infected Nicotiana tabacum K326, Then got three transform materials which containing interference sequence of coat protein gene of TMV or CMV, it has been proofed using molecular method that they have been introduced into Nicotiana tabacum successfully and we also analyzed the mRNA expression differences using real-time quantitative PCR. These data established that the transformed tobacco strains showed resistance to TMV and CMV improved remarkably.
Permeabilized cells containing trehalose synthase from Meiothermus sp. CBS-01 was immobilized by the integration of entrapment in alginate beads with cross-linking and electrostatic self-assembly coating technique. Coating on alginate beads with diazo-resin and poly(styrene sulfonate) can protect the beads from degradation in phosphate buffer, whereas cross-linking with carbodiimide improves the thermostability of trehalose synthase entrapped in alginate beads. By co-immobilization of permeabilized cells using entrapment-cross-linking- coating method, the enzyme recovery was 32%, and the optimum temperature of the enzyme was still 60℃, but the optimum pH was shifted from 6.5 to 7. In batch manner, a maximum conversion yield of 60% trehalose from maltose could be reached by the immobilized cells. Within 4 times repeated use of the immobilized cells, and each time was operated at 50℃ for 24h, more than 50% conversion yield of trehalose could be maintained with less than 20% loss of the enzyme activity.
An important intermediate of deoxyadenosylcobalamin biosynthesis by anaerobic pathway of Propionibacterium freudenreichii was separated from the broth in this paper. It has been qualitatively analyzed by spectrum and ESI MS-GC analysis and further determined as a key intermediate of deoxyadenosylcobalamin: adenosylcobinamide which can be transformed to deoxyadenosylcobalamin when infused 5,6-dimethylbenzimidazole (DMB) to the fermentation medium. It is hope that the intermediate could be a key factor which guides the infusion of DMB and further optimize the fermentation process.
Six hemicellulose-decomposing strains were isolated from the refuse soil samples using bagasse hemicellulose as the sole carbon. The strain with high xylanase activity was screened and identified as Aspergillus sp. HQ3 by analysis of 18S rDNA sequences (97% similarity to Aspergillus sp.) and phenotypic properties. The optimum solid-state fermentation conditions for xylanase production were obtained as: bagasse: bran =7: 3 (W/W), solid: liquid =1: 4 (W/W), urea 0.4%, pH 7.0, fermentation temperature 30 ℃, fermentation time 4d. The activity of xylanase reached 3421 U/g koji under the optimum conditions.
On the base of element and metabolism balancing, the mathematic model of the human-like collagen expression phase with recombinant Escherichia coli BL21 was developed and the unknown parameters in the model were estimated with the method of nonlinear optimization. The model was in agreement with the growth kinetics and the metabolic kinetics, and the key calculated parameters of αh, αp and mx were 1.173 molo C-mol-1, 293.814 molo C-mol-1 and 17.878 molo C-mol-1oh-1 respectively. This model could preferably predict the macroscopic reaction rates, and in the synthesis phase of human-like collagen, the specific growth rate should be controlled at 0.04 h-1 with controlling glucose feeding rate to gain the highest specific production rate of human-like collagen.
The effect of initial substrate concentration on the growth, metabolic activities of Photosynthetic bacteria (PSB) in the process of hydrogen production was studied in the experiment. The experiential formula on the effect of initial substrate concentration on the specific growth rate, specific substrate consumption rate and specific hydrogen production rate of PSB were developed based on Monod model. The predicted results of model which indicated the law of the effect of initial substrate concentration on the growth, metabolic activities and hydrogen production were consistent with the experimental data in growth phase of PSB. The research results indicated that the optimal substrate concentration in the process of growth, metabolism and hydrogen production was 50 mmol/L, when the initial substrate concentration was under or hyper 50 mmol/Lthe activation of PSB was affected, and indicated that initial substrate concentration had little effect on the specific substrate consumption rate.
Objective: To obtain a targeting cationic polymers (KH)20-EGF and examine its capacity for DNA condensation. Method: Prokaryotic expresssion plasmid pET28a-(KH)20-EGF was constructed and transformed into BL21(DE3). The cationic polypeptide was induced by IPTG and purified using a Ni-NTA column. The purified polypeptide was determined by SDS-PAGE and Western blot analysis. The pDNA package ability of the resulted protein was examined by gel retardation assay. Results: The yield of (KH)20-EGF protein was about 300μg/L. The resulted protein was a little larger than expected when examined by SDS-PAGE and Western blot. Gel retardation assays indicated that (KH)20-EGF can retard the pDNA migration. Conclusion: We successfully expressed the non-virus vector (KH)20-EGF and confirmed its capability of DNA condensation.
To amplify microsatellite loci, one fluorescently labeled multiplex-PCR was constructed and optimized using capillary electrophoresis. First, according to the expected length, 9 microsatellite loci were divided into 2 groups , 5 labeled with FAM (Blue) and 4 labeled with HEX (Green). Different fluorescent groups were optimized separately using agar gel electrophoresis. Second, 8 Chinese mitten crabs were was amplified by one set of fluorescently labeled primers（9 loci multiplex-PCR. The Multiplex-PCR products were detected through capillary electrophoresis with ROX500 as size standard and analysised using Genemapper3.5. The results show that capillary electrophoresis has 1 bp precision and can differentiate the main peak and the stutter bands caused by slippage. Uniform PCR products were obtained by adjusting the ratio of primer concentration. Finally, the fundamentals parameters （dNTP concentration、 PCR program and template DNA concentration） were tested one by one. This study showed it was precise, efficient and stable to genotype microsatellite by the detecting the fluorescent multiplex-PCR with capillary electrophoresis.
The concentration of precursors for the production of taxol with Fusarium mairei K178 was optimized by response surface methodology. Firstly, Plackett-Burman design was undertaken to evaluate the effects of eight factors. With statistic regression analysis, the significant factors affecting taxol production were determined as follows: phenylalanine, sodium acetate and sodium benzoate. Box-Behnken design was used to optimize the three critical internal factors mentioned above, and we found out the optimum concentration levels and the relationships among these factors. By quadratic regression model equation with Design-Expert statistic methods, the optimal concentration of the variables were determined as: phenylalanine 2.8mg/L, sodium acetate 3.3g/L, sodium benzoate 31.8mg/L. Under such conditions, the taxol production was increased to 242.6μg/L, which was 15.6% higher than the maximum value in the single factor tests. The experiment values under the optimal conditions agreed with the predicted values, which indicated that the model was proper and effective.
This paper developed a method for the determination of S-Adenosyl-L-methionine in fermentation liquid using the high performance liquid chromatography coupled with UV detector (HPLC-UV). A good linearity was obtained in the range of 0.1~1.0g/L (r = 0.9969) for S-Adenosyl-L-methionine. The average recoveries of S-Adenosyl-L-methionine were 99.89~101.7% and the relative standard deviations were 0.48~1.36%.The method is simple and rapid with reproducibility for the determination of S-Adenosyl-L-methionine in fermention liquid.
Based on the EIAV vector pcPPTWPRE, the plasmids pWCAGP0.8 containing part of the chicken β-actin intron 1 and pWCAGP1.6 containing the full intron were constructed by restriction enzyme digestion.Then the EIAV gene transfer vectors pWCAGP0.8, pWCAGP1.6 and pcPPTWCAG which were previously constructed without the chicken β-actin intron 1 were transfected into HEK293 cells and DF-1 cells using Calcium Phosphate Transfection Kit, respectively. The cells were investigated under fluorescence microscope at 12, 24, 36 and 48h after transfection. Flow-cytometric analysis was carried out at 48h. Results showed that the pcPPTWCAG had the equal EGFP expression level with pWCAGP0.8, while both of them could express EGFP much more efficiently than pWCAGP1.6 in HEK293 cells. In DF-1 cells, the pcPPTWCAG could express the EGFP a little more efficiently than pWCAGP0.8, while both of them could express EGFP much more efficiently than pWCAGP1.6. Results indicated that the chicken β-actin intron 1 could decrease, rather than increase the EGFP expression of EIAV vectors in both HEK293 cells and DF-1 cells.
A nicotine-degrading bacterial strain Z7 was isolated from tobacco growing soil at Zhangjiajie region in Hunan Province. The morphology, physiological and biochemical characteristics of strain Z7 were studied and the results showed that characteristics of this strain were essentially consistent with Agrobacterium radiobacter/tumefaciens. This strain degraded nicotine optimally at 30℃ and initial pH 7. It was able to utilize nicotine as its sole carbon source, and it could degrade 71% of nicotine under the optimized incubation conditions for 48 h. Meanwhile，the color of culture medium turned from yellowy to green and dark green, then it turned to brown. This strain might have potential applications in tobacco industry.
Epitope vaccine is one of the emerging vaccine techniques developing in past decade years. Particularly the advantages of this vaccine on preventing and therapy illness, as cancer and virus, are espacially outstanding. In this article, the most importent elements about the vaccine, namely T/B-epitopes obtainment and identification, vehicles for epitope and vaccine construction, are reviewed. In addition, applications of the vaccine technique in some refractory diseases, such as cancer, virus and pathogen infection, are depicted.
Abscisic acid is one kind of phytohormone with overall physiologic function. It plays an important rule on plants somatic embryogenesis and development. According to the latest literatures, present progress of regulatory role of Abscisic acid in plant somatic embryogenesis is introduced from the following aspects, the influence of ABA on plant somatic embryogenesis, endogenous ABA content change, gene expression, signal transduction and transgenetic expression regulation in the process of plant somatic embryogenesis.
Yeasts were fairly valuable microorganism resources, because it had excellent acid resistant, permeate pressure resistant. Consequently, it was applied for treatment of high concentration organic wastewater, including poisoning and refractory organic pollutants in the wastewater, its treatment ability was prior of acclimated activated sludge system，Moreover, it could adsorption heavy metal ions. And it could transform organic pollutant into non-poisoning, nutrient abundant and delicious single cell protein. With deep of yeasts research and development others relative wastewater treatment technology, wastewater treatment technology would have better and deeper application using yeasts. So it could implementation sustainable development of environment, social and economy.
