Objective: To investigate the establishment of the experimental cell model of MUC5AC and MUC5B high expression induced by human neutrophil elastase (HNE) and the relative mechanism. Methods: Human lung adenocarcinoma A549 cells were cultured and divided into six groups：control group, HNE25nM group, HNE50nM group, HNE100nM group, anti-EGFR antibody+HNE50nM group and AG1478+HNE50nM group. The cell activity was assessed by MTT method. The changes of MUC5AC mRNA and MUC5B mRNA were analyzed by RT-PCR. The protein expression changes of MUC5AC and MUC5B were detected by ELISA, while the protein expression changes of MUC5AC, MUC5B and p-EGFR were observed by Immunofluorescence and confocal laser technology. Results: HNE depressed the cell activity in a dose-dependent manner. The gene transcription and the protein expression of MUC5AC and MUC5B in the HNE-induced groups were significantly higher than those in the control group and the differences were statistically significant (P <0.01). p-EGFR protein expression in the HNE50nM group was much more than that in the control group. MUC5AC mRNA and protein in the anti-EGFR antibody+HNE50nM group and the AG1478+HNE50nM group were lower than those in the HNE50nM group, but MUC5B mRNA and protein had no differences between these groups. Conclusion: Human lung adenocarcinoma A549 cells express MUC5AC and MUC5B simultaneously, and HNE can effectively stimulate the high expression of MUC5AC and MUC5B. The A549 cell with mucin high expression induced by HNE can provide the cell model, which is similar with the environment in vivo and helps the research of airway mucus hypersecretion disease. HNE induces the high expression of MUC5AC via EGFR signal transduction pathway, but MUC5B high expression mechanism is different, which need further study.
Chinese oak silkworm, Antheraea pernyi nuclear polyhedrosis virus (AnpeNPV) as a gene expression vector using A. pernyi culture cell (AnPe cell ) and diapausing pupa has been successfully expressed a foreign gene and produced a great lot protein. This paper compared the expression efficiency of β-galactosidase gene between AnpeNPV with AcMNPV, BmNPV and HycuNPV gene expression vectors in insect culture cells and live insect tissues. The experiments results showed that the maximum β-galactosidase activity were AnpeNPV cells 40.9 units/ml (TC-100 medium, FBS 10%) and 59.9 units/ml (SF-900 medium II) in AnPe cells, AcMNPV 72.4 units/ml (TC-100, FBS 10%) and 66.4 units/ml (SF-900 II) in Sf9 cells, 326 units/ml in High5 cells (EX-405 medium), BmNPV 15.1 units/ml in Bm4 cells (TC-100, FBS 10%), HycuNPV 68.6 units/ml in SpIm cells (SF-900 II) in 5 × 105 cells, individually. In live insect tissues, the maximum β- galactosidase activity were A. pernyi female pupae 14.3 units/g, male pupae 11.7 units/g, Bombyx mori 5th instar larvae 10.1 units/g. In culture cells, the β-galactosidase level of AnpeNPV was similar to AcMNPV with exception of using High5 cells and to HycuNPV, better to BmNPV. In live insect tissues, the β-galactosidase level of AnpeNPV was similar (male pupa) or significantly higher (female pupa) than BmNPV. AnpeNPV gene expression vector exhibited similar expression efficiency to other NPV vectors in both cultured cells or live insect tissue. Compared to other expression system. The diapausing pupa is easily operated, and suitable for large amout production.
Abstract Human single chain interleukin-12（hscIL-12）of bioactivity was expressed in methylotrophic yeast Pichia Pastoris expression system , hscIL-12 gene was amplified from plasmid pBI121-IL-12 by PCR. After digested by restricted enzyme, gene of interest was cloned into the yeast vector pPIC9K and obtained recombinant expression vector pPIC9K-hscIL-12. The pPIC9K-hscIL-12 was linearized with SacⅠ and then transformed into the Pichia Pastoris GS115 by PEG1000. Recombinant strains were screened by G418 resistant, and further confirmed by colony PCR. The hscIL-12 was induced to express in yeast by methano1. Western Blot analysis showed that the relative molecular weight of the expressed product was about 70kDa, the expression protein could bind to IL-12 monoclonal antibody specifically. Quantitative analysis showed that the target protein was in a level of 26% of the total protein of the culture supernatant，with a yield of 40mg/L. Bioactivity assay showed that the recombinant hscIL-12 could stimulate the lymphocyte proliferation. The successful expression of the rhscIL-12 in Pichia Pastoris can be potentially used in cancer gene therapy.
In plants, oxidative stress is one of the major causes of damage as a result of various environmental stresses and it is primarily due to the excessive accumulation of reactive oxygen species. To develop transgenic potato plants with enhanced tolerance to environmental stress, transgenic potato plants (Solanum tuberosum L. cv. Atlantic) expressing the Cu/ZnSOD and APX genes in chloroplasts were generated under the control of the oxidative stress-inducible promoter. To investigate oxidative stress tolerance, transgenic plants were evaluated at the level of leaf discs and plantlets after methyl viologen (MV) and salt treatment. Leaf discs from transgenic potato plants showed 13% less membrane damage compared to non-transgenic (NT) plants suffering 10 μΜ MV treatment of 48 h, and showed 1.6-fold higher chlorophyll contents than those of NT plants at 1.0 M NaCl treatment (31% vs. 19%). In addition, transgenic potato plants maintained higher rooting rates (75%) during 100 mM NaCl treatment than those (12%) from NT plants. Moreover, the tolerance to salt stress in transgenic plants was consistent to increased transcript levels and higher activities of SOD and APX compared to NT plants. These results suggest that expression of Cu/ZnSOD and APX in chloroplasts could be used in plants to enhance the tolerance to environmental stresses.
To explore the expression and function of antibacterial peptide CM4, the expression and biological activity of recombinant fusion protein CM4-hsBAFF were studied. The human soluble B lymphocyte stimulator activing factor (hsBAFF) gene was fused to the sequence encoding CM4 to construct an expression vector pET28a (+)/CM4-hsBAFF. The recombinant protein was high expression of soluble recombinant protein in Escherichia coli cells, and existed in the supernatant after sonication. Recombinant fusion protein which was purified through size-exclusion chromatography was identified by SDS-PAGE and Western blot analysis. SDS-PAGE and Western blot indicated that recombinant protein was secreted as a protein of around 22.0 KDa and can be specially recognized anti-hsBAFF antibody. The recombinant protein can be expressed and displays antimicrobial activity.
Filamentous fungus Trichoderma harzianum Th-33 was successfully transformed with Agrobacterium tumefaciens EHA105 for random integration of transforming DNA(T-DNA). Co-cultivation of T.harzianum Th-33 conidia with A. tumefaciens EHA105 in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency(about 45~100 transformants per 106 conidia). T-DNA insertional library of T.harzianum Th-33 mediated by A. tumefaciens was constructed, which consisted of more than 4000 transformants. Twenty-four randomly selected resistant clones were proved to be stable through five generations mitotic cell division. The integration of the hph gene into T. harzianum genome was determined by PCR and southern blot analysis. The results showed that T-DNA was inserted into all the transformants with random and single copy mainly. These suggest that A .tumefaciens mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene clone for T. harzianum .
A novel biosensor for aflatoxin B1 detecting has been reported in this paper. The biosensor electrode for AFB1 detecting was assembled by immobilized aflatoxin-oxidoreductase using open-ended multi-walled carbon nanotubes as matrix. Its linear range was between 0.16μM and 3.2μM. And if the specific anti-aflatoxin B1 antibody and aflatoxin oxidoreductase were both immobilized on the electrode with Multi-Walled carbon nanotubes, the detection limit of the modified electrode could be 16 nM with a 10 times improved sensitivity. The aflatoxin enzyme biosensor assembled this way strode one step forward its practical application.
According to the amino acid sequence of monellin and the bias in coden choice in Escheria coli，a single chain monellin gene was synthesized and subcloned into a Escheria coli expression vector pET-28a. The recombined plasmid pET-28a-Mon was then transformed into Escheria coli BL21(DE3).Lactose was used as an inducer instead of IPTG.Through proper optimization of induction conditions，an expression level 33.09% of total cellular protein was achieved.The expression level of recombinant protein induced by lactose was basically the same as that induced by IPTG.The result indicated that lactose could be used an promising inducer in the production of recombined Monellin.
Objective: to evaluate biological safety of HAP/AZ31B as implanted material. Methods: HAP/AZ31B biomaterial was prepared by cathodic electrodeposition with constant voltage technique, and then biocompatibility was assessed. The corrosion of surface was detected by spectrum analysis. Results: HAP/AZ31B had no mutagenesis and toxic effect. Serious inflammatory reaction was not induced. The corrosion velocity of HAP/AZ31B was slower. The micronucleus frequency and hemolysis of HAP/AZ31B leaching liquor was 4.4‰ and 0.25%, respectively. Conclusion: HAP/AZ31B had excellent biological safety and biocompatibility, which would be a new degradable and biomedical material.
Saccharomyces cerevisiae was used as a new type of biosorbent for the removal of copper. Various experimental parameters (e.g. equilibrium pH, contact time, Cu2+ concentration) were investigated. The biosorption of Cu2+ by saccharomyces cerevisiae is a quick process which only need 4-6 hours to reach the equilibrium, and the optimum pH is 5.It is indicated that Langmuir isotherm sorption model is suitable for the isotherm adsorption of copper. Sulfonate, carboxyl and amine groups were present in the biosorbent, which was confirmed by FTIR and proton-binding model. Chemical sorption was dominated in the binding of its molecules. Different functional groups were ionized in different pH conditions and played a pronounced role in the sorption.
A rapid extraction method for genomic DNA was established by using silica-modified magnetic nanoparticles (MNPs) prepared by our research group, protocol and buffer solutions designed by ourself. In this method, silica MNP was used as a solid carrier for absorption, guanidine hydrochloride, β-mercaptoethanol and SDS were employed as major contents of lysis binding solution. Extraction results appeared that 2～3 μg genomic DNA can be obtained from 100μL mouse whole blood or cultured cells by the method established in this study, and the OD260/OD280 is 1.8 ± 0.05. In addition, the purified DNA can be available for the down stream experiments such as enzyme digestion and PCR. Extraction time in this method only need about 12min, also there is no proteinase K digestion step and centrifugation process. Consequently, it is a rapid, simple and high efficient extraction method applicable to universal laboratories.
The process of extracting exopolysaccharide(REPS) from Rhizobium sp. N613 was optimized in this study. The optimized conditions of extracting REPS were obtained by Response Surface Methodology. They are pH value 5.9, concentration of ethanol 74% and precipitation time 16.5 h, respectively. Under those conditions, the extraction yield was 9.28±0.06 g · L -1, the purity was up to 97% and the extraction rate was 93.6%. At the same time, the thesis carried out the test of the viscosity、 acute toxicity and accumulative toxicity of REPS. It is showed that REPS has high viscosity and nontoxicity. So it is more valuable for the application for food and medicine.
Acidithiobacillus ferrooxidans are able to synthesize intra-cellular electron-dense magnetite，which makes possible that we could obtain bio-nano-magnetic particles by cultivating Acidithiobacillus ferrooxidans. In order to isolate the strain which has the capacity to produce more magnetic particles, the solid-plate magnetophoresis method was firstly created. After isolation using the method, the rate of the cells which contain intra-cellular magnetic particles was increased from 30% to more than 90%, in addition, after isolation each cell possessed 2-5 magnetic particles which disperse in cells. The isolated cells are able to orient and migrate to the magnet in artificial magnetic field but could not orient swimming only under the geomagnetic field. Magnetosomes produced by Acidithiobacillus ferrooxidans were range from 40nm to 90 nm according to the results of TEM. Energy dispersive X-ray analysis indicated that extracted magnetic particles consisted of oxygen and iron. The results show that some Acidithiobacillus ferrooxidans cells have weak magnetotaxis and they could be able to be separated by solid-plate magnetophoresis method from others. With the development of this new isolation method, it is possible that we could do deeper research to generate a comprehensive description of the mechanism that how Acidithiobacillus ferrooxidans synthesize the magnetic particles.
In order to investigate the effects of Osx on Osteoblast proliferation and differentiation, recombinant Osx-expressing adenovirus were constructed. Osx-coding sequence was cloned into adenovirus vector, which was transfected to 293A for packaging, primarily cultured mouse osteoblasts were infected with the virus, alizarin red staining was used for mineralization analysis, realtime quantitative RT-PCR was used for differentiation assay, cell cycle was checked using FACS. Results show that ① Virus titer was about 2×109/ml，most optimal multiplicity of infection was 50, ② Overexpression of Osx did not promote osteoblast mineralization, ③ 1d,3d and 6d after overexpression of Osx upregulated the transcription level of Osteocalcin, BSP and collagen Ⅰsignificantly(p<0.01), ④ Osx promoted osteoblast proliferation(p<0.01). Overexpression of Osx in osteoblast promotes proliferation of osteoblast, and regulates osteoblast differentiation to a certain degree.
Objective:To study main biochemical characterization of a group of earthworm Desoxyribonucleases(EWDs). Methods:SDS-PAGE and MADLT-TOF ,enzyme activity detecting method were used to determine EWD1，EWD2, EWD3. Results:The molecular weights of EWD1, EWD2, EWD3 were 157.2kDa、69.1kDa and 63.5kDa respectively. Enzymological studies showed Kms of three EWDs were 1.58, 3.95 and 1.52 mg /ml. These 3 Dnases are sensitive to temperature, they do not display activity when the temperature rises to 55 degree. The optimal pH was between 4.0-5.6. Mn2＋、Ca2＋ are inhibiters for their activities, and Mg2+ had impact on EWD3 activities. Conclusion:Their MWs are apparently larger than DNaseⅠand DNaseⅡ. Temperature stabilities, pH stabilities, stimulant and antagonist studies were conducted on EWDs, the parameters of these effective factors were different from that of present Dnases. The above indicated that EWDs found in Lumbricus Bimastus had special enzymological characters and were new DNases, which were different from DnaseⅠand DNase II their catalytic mechanisms and classification wait to be studied and sorted.
To improve the laccase fermentation productivity of Coriolus vericolor, a novel method for laccase fermentation production was proposed in a three-phase bubble column bioreactor with the mycelial pellets under repeated batch process. Under the optimum laccase production conditions, the effects of the added ascorbic acid concentration on laccase activity were investigated and laccase was cultured by adding ascorbic acid in culture medium under repeated batch process. When 1.50mmol/L ascorbic acid was added to the medium, the laccase activity of fermentation broth reached 1557.9 U/mL, which was 1.6 times higher than that of the control (no ascorbic acidwas added). Under the certain condition, laccase was produced repeatedly in 10 batches for 25 days in the repeated batch culture in the bubble column. The maximum laccase activity was 1919.6 U/ml in the fifth batch. In each batch ,the maximum laccase activity were all more than 1000 U/ml. Laccase produced by the method decolorizes indigo blue markedly. The decolorization rate reached 96.7% with 1-hydroxybenzotriazole（HBT） as mediator for 40 min, HBT concentration 0.10% and laccase dosage 100 U/l. The performance of the three-phase bubble column bioreactor is stable, and the mycelial pellets can be used repeatedly to effectively yield laccase by the method in large scale.
Fibroblast activation protein α (FAPα), a surface antigen especially expressed on carcinoma associated fibroblasts (CAFs), is involved in tumor cells growth, adhesion, invasion and metastasis for its dipeptidyl peptidase and collagenase activities. Over expression of FAPα on CAFs and its special endopeptidase activity represent a potential target for diagnosis and treatment of various carcinomas, making it feasible to be applied in oncogene therapy and immunotherapy of targeting tumor cells. This review focuses on the relationship between FAPα and tumor development, and summarizes a series of studies involving FAPα structure, enzyme activities as well as clinical diagnosis and therapy of malignant tumors.
Antihypertensive peptides (AHPs) have good effect of decreasing blood pressure, also have low adverse effects and toxicity. Now it has been received great interests in gene engineering. This paper briefly reviewed the necessity of construction gene-engineering strain which expressing AHPs and other small peptides. Discussed the strategys and theoretical foundation of chosen peptides, construction tandem repeats, chosen expression vectors during the gene engineering constructions, mainly introduced the advantages and disadvantages of different types of expressing vectors. The purpose of this paper is to providing a reference for the future construction gene-engineering strain which expressing AHPs and conveniencing the design of this kind of experiment.
In plant genetic engineering, selectable marker is needed to distinguish transformant. As the commercialization of transgenic plants, people are more and more paying close attention to their safety, among which refers to the safety of selectable marker. In order to increase the safety of transgenic plants, Biologists began to search for biosafe selectable marker. Here we overview current research progress of taking key enzyme in metabolic pathways，which include glycometabolism, amino acid metabolism, hormone metabolism ,nucleotide metabolism and protein metabolism et cetera, as selectable marker in transgenic plants.
Abstract This review focuses on the carboxylestereases from hyperthermophiles. Most of them are similar with the hormone-sensitive lipase (HSL) family in structure. Both the carboxylestereases and HSL belong to α/β hydrolase. And their structures become more compact and tenacious compared with mesophilic esterases. This special structure enhances their thermostability. The review introduces the effects of temperature and organic solvents on the catalytic efficiency and enantioselectivity of the carboxylesterases. Generally the optimum substrate of the enzyme is medium chain p-nitrophenyl，and the presence of a GGGX motif in these carboxylesterases suggest the enzyme has the ability to hydrolyze tertiary alcohol esters. Because of their characteristic, the carboxylesterases could be used in various aspects，especially in separation of racemic ester.
