To construct a expression vector induction by RU486 (mifepristone) and confirm the regulatable effect in vitro.The PRS plasmid vector encoding a GLP65 chimeric regulator and GAL4 hybrid promoter was modified with molecular biological methods. The BGHpolyA fragment was amplified by PCR technique and introduced several enzyme cutting sites. A hCMV promoter replaced the TTR promoter to control the GLP65 regulator, and luciferase reporter gene was inserted into downstream of the GAL4 promoter. To minimize any potential interference, the two transcriptional elements were spaced with a 1.2kb chicken beta-globin insulator. The recombination vector was identified by different restriction endonuclease reactions, sequencing analysis and PCR assay. Dual-Luciferase Reporter Assay was used to demonstrate the activation of this regulatable vector after cotransfection with pRL-TK in HEK293 cells．Forty-eight hours after transfection, cells were harvested to determine luciferase activity by illuminometer. In the presence of RU486 a 40-fold maximum activation of the luciferase reporter gene was observed in cultured cells, whereas in the absence of RU486, no significant activation was observed. There was a positive correlation between the luciferase activation and RU486 concentration in a definite range. The results showed that the RU486-inducible regulatory vector was successfully constructed, which can be used to regulate the expression level of genes of interest in appropriate time by the inducer RU486. This inducible expression vector provides a platform for the research of gene regulation and gene therapy.
To explore the technics for high expression, purification, quality control of recombinant human erythropoietin and IgG Fc fusion protein, rhEPO-Fc fusion protein expressed by CHO cells in serum-free medium was collected and purified with a three-step purification process-affinity chromatography, ion exchange chromatography and molecular sieve chromatography; and its quality was controlled by protein concentration、SDS-PAGE、Western-blot、HPLC、ELISA and IEF, et al. The research findings are that the expression output of rhEPO-Fc was highly reached 2g/L, the purify rate was over 45%, its purify was over 98%, its relative molecular weight is about 60KD, t1/2 in vivo of rhEPO-Fc is over 38h, western blot analysis showed rhEPO-Fc has nature antigenicity. The expression output and purify rate of the produce technics is high, quality inspection methods are stability and reliable, which could be fit for in large-scale production of rhEPO-Fc.
0bjective To investigate the protective effects of MaFGF on the injury of cultured renal tubular epithelial cells induced by DDP. Methods These primary cells were inoculated into 96 well plates. (1)After 72h culture，DDP with different concentration was added，and MaFGF with different concentration was added further as the experimental group after 12h of the DDP addition. WST-8 assays were performed to test the cell viability after 36h treated by MaFGF.（2）The injury model was induced by DDP. And the protective effect of MaFGF on the injured renal tubular epithelial cells was observed. Results (1)The concentration of DDP that inhibited 50% cell growth(IC50) was increased by MaFGF；(2)The biochemical and zymological results between the DDP group and the control group were different significantly. However，with the addition of MaFGF， the differences of SOD、GSH-Px enzyme activity between the two groups were insignificant，except for MDA and NO. Conclusion MaFGF could exert obvious protective effects on the renal tubular epithelial cells damaged by DDP
Objective Evaluation the application value of Fatty acid methyl ester (FAME) method in strain identification and relationship judgment. Methods 194 V. cholerae strains belonging to the two main pathogenic seragroups, El Tor isolated since 1961 and O139 since 1992 in China, were selected and fatty acid methyl ester were extrcted. Microbial identification system of MIDI company was used to analysis the data. Results Thirteen fatty acids were common components contained in all of the tested V. cholerae strains. The coincidence of special judgment was 88.6%. There was no obvious separable group in 2-D plot analysis. Conclusion FAME method is useful in rapid identification of Vibrionaceae strains and in assistant field isolation of V. cholerae. It can be used to judge the relationships of strains in small outbreaks, but it cannot differential the subgroups of V. cholerae.
Augmenter of liver regeneration (ALR) is a kind of hepatocyte growth factor .To study the expression, purification and bioactivity of human augmenter of liver regeneration (rhALR) in Pichia Pastoris, the expression plasmid pPICZαA- ALR was constructed and transformed into P. Pastoris by the method of electroporation transformation. Induced with 0.5% methanol, the 30 kD protein in the culture supernatant of recombinant P. Pastoris was confirmed to be rhALR by SDS-PAGE and Western blot. Quantitative analysis showed that the target protein was in a level of 66% of the total protein of the culture supernatant, with a yield of 40mg/L.we had performed DEAE anion exchange chromatography two times with excessive and regular adsorption quantity consecutively, and then the rhALR above 95% purity and 52% protein recovery could be obtained by G75 molecular sieve chromatography at last step. The bioactivity assay of the purified product showed that rhALR could stimulate the proliferation of HepG2, SMMC-7721 and NIH-3T3 in vitro.
Study the function of p21-activated kinase 2 (PAK2) in Xenopus oocyte maturation. Inject PAK2-N teminal (PAK2-NT) mRNA to Xenopus oocyte,and observe germinal vesicle breakdown by fluorescence microscope. To further observe the changes of F-actin and spindle, confocal microscopy with time-lapse was employed.Compared with the controls, injection of PAK2-NT mRNA caused no visible changes in oocyte morphology nor affected progesterone-induced germinal vesicle breakdown,but block cytokinesis and polar body formation. Maybe PAK2 involves in Xenopus oocytes maturation.
The third component of human complement (C3) is an important participant in immune surveillance and immune regulation. There are many binding-sites on the surface of human C3 molecule, especially the binding-sites to complement receptor on blood cell, which induce the regulation of complement, phagocytes, and even bacteriolysis of heterogenous pathogens. So we fused the genes of two C3 binding-sites which adhered to complement receptorⅠ (CRⅠ) and complement receptorⅢ (CRⅢ) by overlap extension PCR. Bactericidal-permeability increasing protein (BPI) is a kind of cation proteins separated from heterophil granulocyte which shows strong attachment and disinfection on G- bacteria, even some fungi and protozoon, especially in total blood, plasma. In this research, the active part of BPI, rBPI was obtained by PCR. Then rBPIs was connected to the genes of human complement C3 binding-sites by gene splicing in target to fusion protein of C3-BPI active part, which was named CB. CB fusion protein was supposed to has the function of killing exogenous pathogens and introducing adherence of red blood cell, phagocyte, etc, which would lead to the fast clearance of pathogens in blood. Then pET28-CB, a prokaryote expression vector of CB was constructed and efficiently expressed in Escherichia coli BL21(DE3). The result of western blot showed the target protein had the activity of combination with C3 antibody, Highly concentrated purified protein was obtained after denaturization and renaturation of extracted inclusion protein. The expression and purification of the CB fusion protein was suitable for further analysis of its function and application.
The development of immunotoxin DT386-GMCSF, a fusion protein which bears the N-terminal 386 amino acids of diphtheria toxin and human granulocyte-macrophage colony-stimulating factor (GM-CSF) and targets the GM-CSF receptor (GM-CSFR), has provided a promising alternative therapy to the acute myeloid leukemia (AML). However, the poor expression of the protein in E.coli is still a bottleneck which limits the industrial production. To identify the critical down-regulating factors on the expression of DT386-GMCSF, a series of truncated mutants of DT386-GMCSF at the C-terminal of GM-CSF were generated and expressed in E. coli. The results showed that the encoding sequences for the L114 of the GM-CSF dramatically impact the expression of DT386-GMCSF. On this basis, a serial of mutants integrating amino acid substitutes were generated. The results revealed that the expression level of the mutant DF123GVT, which harbors the amino acids 1-123 of GM-CSF and the L114L115V116 was substituted with G114V115T116, was evidently higher than that of the DT386-GMCSF, whereas the specific cytotoxicity to blast recovered from mice injected with HL60, a cell line highly expresses the GM-CSFR, was similar. These results provide an important basis for the future development of the immunotoxins targeting the GM-CSFR.
Abstract: To explore the effect of C-terminal region residues on the substrate specificity of a novel cyclic imide hydrolase (CIH), a recombinant cyclic imide hydrolase (CIH293) and its mutant enzymes deleted or substituted at C-terminus (CIH291, CIH290, KK292-293EE) were prepared by gene cloning. Their substrate specificity and kinetic parameters were analyzed by both the spectrophotometric assay and high-performance liquid chromatography. Results show that the substrate specificity of mutant enzymes was not obviously changed, but slightly low for the substrate affinity, compared with the wild-type enzyme, CIH293.
To investigatethe feasibility to produce crocetin in tobacco plants. In this study, the coding region of zeaxanthin cleavage dioxygenase (CsZCD) gene from crocus sativus L. was inserted into the downstream of the cauliflower mosaic virus (CaMV) 35S promoter of a binary vector pBI121, and integrated into the genome of Nicotiana tabacum L. Twenty-one transgenic lines were identified by genomic southern blotting analysis. Western blotting and HPLC analysis of the leaf extracts of transgenic tobacco showed that crocetin was produced in CsZCD gene-transformed plants, while no crocetin was found in the untransformed tobacco.
Monoclonal antibody isotyping micro–well protein array was designed and established. Twelve monoclonal antibodies and two polyclonal antibodies were isotyped by the isotyping protein array and ELISA respectively. The results showed that there was no difference between protein array method and ELISA in the application of isotyping monoclonal and polyclonal antibodies. And fewer sample and materials were used and the efficiency was improved in protein array method compared with ELISA. As a result, the antibody isotyping protein array could be used as a sensitive, rapid, directional and economical tool applying in high throughput monoclonal antibody generation.
In order to improve the accuracy and sensitivity in detecting of chitinase isozymes from Trichoderma. spp, a rapid sensitive method is established. It was employed to detect chitinase isozymes from Trichoderma viride LTR-2 through native gel electrophoresis, denatured gel electrophoresis and in situ gel electrophoresis with Calcofluor white M2R. Two chitinase isozymes native bands CHI65 and CHI42 were revealed in solid plate containing chitin and Calcofluor white M2R with native gel electrophoresis after chitin grown culture was concentrated 5 times. The two bands showed a smear instead of well-defined band with in situ gel electrophoresis after 20 times concentration. While only CHI65 chitinase was found with denatured gel electrophoresis after 10 times concentration. The molecular weight of CHI65 and CHI42 was separately about 65KDa and 42KDa. As a result, it was suitable for detecting chitinase isozymes through active gel electrophoresis with Calcofluor white M2R. The result showed that it was effective for differentiation among chitinase isozymes when total chitinase loading amount was 0.47U.
High quality of total RNA from soybean leaves suitable for high efficient construction of cDNA library and gene cloning based on SMART strategy was isolated by the improved Acidic-Phenolic method. Five full length genes of isoflavonoid biosynthetic pathway were successfully and quickly amplified by PCR method with the original library cDNA dilution as templates. Comparing with traditional method of cloning genes from DNA or RNA, or isolating genes from cDNA plasmid hosted bacteria, this method can obtain full-length multi-gene more quickly and simply.
Lipase extraction of Cryytococcus neoformans using aqueous two-phase system has been studied in this paper.The polyethylene glycol(PEG)/salt system has been systematicly researched,the system of PEG/（NH4）2SO4 was better to separate this lipase.Many factors which affecting the ability of phase separation and extraction has been investigated. In this case, PEG 15%,（NH4）2SO4 22.5%, pH8.0 without NaCl was the optimized condition,and the partition coefficient and purification multiple was 6.8 and 7.5 respectively.Its specific activity got to 40.3 U/mg protein.
The expression vector pGg-gfp containing gpd-Gf (615bp) promoter fragment from Grifola frondosa was constructed for detecting its functional activity by the fusion of promoter fragment to the reporter gfp gene. Co-transformation of plasmid pGg-gfp and plasmid pCc1001 which harbors the complementary gene trp1 was conducted by the PEG-mediated protoplast transformation of the oidia of LT2, a tryptophan auxotrophic strain of Coprinus cinereus. The putative trp+ transformants were obtained from the selective regeneration medium and confirmed by PCR detection, Southern blotting and green fluorescence analysis. The results indicated that gfp gene was integrated into the gemone of LT2 strain. And the gfp-Gf promoter could drive the gfp gene expression in Coprinus cinereus. Strong green fluorescence acitivity was observed in the mycilia of Coprinus cinereus transformants under the fluorescent electronic microscope and laser scanning confocal microscope.
Bacillus subtilis glutamyl-tRNA reductase encoded by hemA gene as rate-limitation enzyme involved in biosynthesis of 5-ALA that is the precursor of the biosynthesis of terapyrroles as well as an important functional molecular in medical and agricultural application. The hemA gene was polymerase chain reaction amplified from B.subtilis genome DNA and cloned into the expression vector pET28a.The hemA gene was High-level expressed in Escherichia coli BL21(DE3) when induced by IPTG, in which the recombinant protein make up 20% of total soluble protein. We also obtained the purified protein of HemA through the Ni2+-NAT affinity chromatography resin. Biosynthesis level of 5-ALA was enhanced responding to expression of the recombinant protein. The production of 5-ALA reached 40.2mg/L. The catabolite metabolism of 5-ALA porphyrins was also accelerated.
Response surface analysis(central composite design of uniform precision) of SAS 9.1.3 system was applied to optimize the prime four factors(the ratio of stuff and water, the quantity of enzyme ,the time of fermentation and the quantity of inoculation)for solid fermentation of soybean meal in order to researching the contributiveness of factors and their interactions to the degree of hydrolyzation of soybean protein. Through simulating the predictive model of the quadratic polynomial regression，the functional connection of the factors and response ,that is the equation of polynomial regression，was established and the value of optimization was obtained，namely the ratio of stuff and water is 1:1.00；the quantity of enzyme is 2.55%；the time of fermentation is 65 hours；the quantity of inoculation is 1.00%. Under the optimum level determined，the degree of hydrolyzation is 13.3% and is increased by 56%.
The expression of sLea/x which correlates with conventional histopathologic parameters serves as a useful indicator for the prognosis of metastatic disease. The bindings between sLea/x and their common ligand E-selectin initiate hematogenous metastasis of cancer. Certain bioactive conformation is crucial for the interaction between sLea/x and their ligands. Thus, a new class of compounds that mimic the structures of sLea/x can potently inhibit not only their functional bindings to selectins, but also the metastasis of cancer. This review is mainly on the relationship between sLea/x and hematogenous metastasis of cancer and the design of drugs that mimic the structures of sLea/x.
Abstract: The molecular delivery vectors used in gene therapy need provide the features of safety，stability, efficiency and capacity. The current studies on the structure and action mechanism of pRNA, a packaging RNA of phageΦ29, showed that pRNA with multiple binding sites can through cell membrane easily and escort exogenous molecules to target cell, without inducing immune reaction. As an ideal nano-scale gene therapy vehicle, pRNA presents a promising application in delivering multiple therapeutic components to detect and treat human diseases.
As an application of peptides and protein drug- delivery system, liposome is a kind of promising, broadly applicable drug-delivery system. It can control the drug releasing、deduce the drug toxicity、have the target proerty and so on. In this article , the mainly development including the methods of preparation, new liposomes, administeration of peptides and protein liposomes, was reviewed, according to the references of the development of peptides and protein liposomes in recent 10 years, Furthermore, the updated progresses of liposome of peptides and protein drugs were described, in order to provide some references to the research on liposome in biological area. Some problems in this drug- delivery system were also put forward. Although liposome has made considerable progress and development in the biological area, there are many technological tough problems have not been tackled. The further research on peptides and protein liposomes is also needed.
Bone marrow mesenchymal stem cells (MSCs), multipotent stem cells, can replicate as undifferentiated cells and have the potential to differentiate into different lineages of mesenchymal tissues, including bone, cartilage，endothelial, neural, smooth muscle, skeletal myoblasts, and cardiac myocyte cells. The ischemia-induced death of cardiomyocytes results in scar formation and reduced contractility of the ventricle. Several preclinical and clinical studies have supported the notion that MSCs therapy may be used for cardiac regeneration.When transplanted into the infracted heart, MSCs prevent deleterious remodeling and improve recovery, but the mechanism is not clear. In this work,we review evidence and new prospects that support the use of MSCs in cardiomyoplasty.
Abstract The combination of biology and micro/nano fabrication technology is becoming a new trend, DNA molecules have many characteristics, such as good thermal stability, linear geometry structure and mechanical rigidity, which can be used as ideal templates for nanowire fabrication. By utilizing metallization technology, DNA can make up conductive metal nanowires and nanoclusters, which provides a possibility of constructing nano-devices by metallized DNA molecules. In this article, firstly, we introduce DNA metallization principles. Secondly, we discuss several novel DNA metallization processes, including whole DNA metallization, selective DNA metallization, ionic surface masking decreasing nonspecific DNA metallization and evaporation DNA metallization. Finally, by self-assembly and self-recognition techniques, metallized DNA can be used to build up electronic circuits and used as the metal deposition templates to construct bio-nano devices.
Hormones are the main materials which regulate the plant growth and the secondary metabolites formation of plant. In this paper, the effects of hormones on cell biomass and metabolites content and the application progress of honrmones in plant cell suspension culture is reviewed. It covers the influence of exogenous hormones' categories, concentration and proportion on cell biomass and metabolites content in plant cell suspension culture, the developmental course of the endogenous hormones determination, the changes of endogenous hormones content and its effects on the cell biomass and metabolites content, the relation of interaction between exogenous hormones and endogenous hormones, the study on the effect of new hormone varieties on the culture.
The microbial oxidation of ferrous iron to ferric from by Acidithiobacillus ferrooxidans is a significant step in the bioleaching of sulphide minerals, removal of hydrogen sulphide from gaseous effluents, treatment of acid mine drainage and heavy metal in sludge. Over the years, much attention has been paid to this organism and the process it catalyses. However, the slow oxidation rate of ferrous iron and the poor stability restricted its commercial application by this time. So, immobilization of the cell as well as bioreactor would be the key point for industrial applications of this technology. In the present article a brief overview and the future development of the applications of the bacteria were provided, at the same time the efficiencies of various immobilization materials as well as the different bioreactor systems used to date were discussed and analyzed.
Biotechnology is a strategic development area, and also a key competitive technology to wining advantage in bioeconomic era globally. From online Science Citation Index (SCI) database we performed nine critical technology bibliometrics recombinant protein, monoclonal antibody, vaccine, stem cell, genetic therapy, tissue engineering, biochips, genomics, proteomics by country for benchmarking. Meanwhile, Industry chain gap was also discussed combined with biotechnology product and industry status ,to find gaps between China and developed country and the position of China in global biotechnology landscape. Statistics show American wined Absolute Advantage in all areas , China had some comparative capability in biochips ,bioengineering and Omics area, and will be a follower in a long period of time in biotechnology research and development.
