Abstract:To develop a GFP transgenic cell model under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted. Synthetic oligonucleotide ARE motif was annealed and purified then inserted into pTK-GFP to construct the vector of pARE-TK-GFP. The TK and ARE-TK fragments were amplified by PCR and cloned into pEGFP-N₁ to reconstruct eukaryotic expression vectors of pTK-GFP/Neo and pARE-TK-GFP/Neo. They were transfected into HepG^{2 cells and clones resistant G418 were isolated. PDTC and Lentinan were used to induce the cell levels of GFP and the fluorescence was measured using a fluorescence plate reader. The results showed that the induced level of GFP is significantly increased and have dose-dependeny in a certain range. This findings indicated that such a cell model offered a potential platform for preliminary screening of all kinds of natural or synthetic chemopreventive agents.}

Abstract:To develop a GFP transgenic cell model under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted. Synthetic oligonucleotide ARE motif was annealed and purified then inserted into pTK-GFP to construct the vector of pARE-TK-GFP. The TK and ARE-TK fragments were amplified by PCR and cloned into pEGFP-N₁ to reconstruct eukaryotic expression vectors of pTK-GFP/Neo and pARE-TK-GFP/Neo. They were transfected into HepG^{2 cells and clones resistant G418 were isolated. PDTC and Lentinan were used to induce the cell levels of GFP and the fluorescence was measured using a fluorescence plate reader. The results showed that the induced level of GFP is significantly increased and have dose-dependeny in a certain range. This findings indicated that such a cell model offered a potential platform for preliminary screening of all kinds of natural or synthetic chemopreventive agents.}

Abstract:White-rot fungus manganese peroxidase (MnP) oxidizes a wide range of substrates, rendering it an interesting enzyme for potential applications. The stability of MnP can be improved by immobilization. With sodium alginate, gelatin, or chitosan as a carrier, and glutaraldehyde as the crosslinking agent，MnP was co-immobilized using the embed-crosslinked method and the adsorb-crosslinked method. The immobilization conditions and the partial properties of the three immobilized enzymes were investigated. When compared with the free enzyme, the optimum pH values and the temperatures of the three immobilized MnPs carried by alginate, gelatin, and chitosan were respectively shifted from 7.0 to 5.0, 5.0, 3.0 and from 35℃ to 75℃, 55℃, 75℃.The thermostabilities of the three immobilized MnPs were considerably better than that of the native enzyme. The chitosan-immobilized enzyme was stable in the wide range of pH 2.2 to pH 11. The enzyme activities of the three immobilized MnPs decreased by less than 5% even after repeated use for 6～9 times. The ability of decolorizing azo dyes in static and shaky situation by gelatin-immobilized MnP approached to the free enzyme, and there was no loss of enzyme activity during 2 repeated batch reactions.

Abstract:A lipase from Aspergillus niger F044 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography and Sephadex G-75 gel filtration chromatography. This purification protocol resulted in a 73.71-fold purification of lipase with 33.99% final yield, and the relative molecular weight of the enzyme was determined to be approximately 35～40kD using SDS-PAGE. The optimum pH and temperature for lipolytic activity of the lipase was 7.0 and 45℃, respectively. It was extremely stable at 60℃ and retained 98.70% of its original activity for 30min. The stability declined rapidly as soon as the temperature rose over 65℃. The lipase was highly stable in the pH range from 2.0 to 9.0 for 4h. Ca²⁺ and Mg²⁺ ions stimulated lipolytic activity, whereas Mn²⁺, Fe²⁺ and Zn²⁺ ions caused inhibition. The values of K_m and V_max calculated from the Lineweaver-Burk plot using pNPP as hydrolysis substrate were 7.37mmol/L and 25.91μmol/(min·mg), respectively. The N-terminal sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln, which is highly homogeneity with that of lipase, as reported by Torossian.

Abstract:Pectinases are mainly used in the food industry to clarify fruit juices and wine, improve oil extraction, remove the peel from the citrus fruit, increase the firmness of some fruits and degum fibres. The filamentous fungus Aspergillus oryzae, used for the production of traditional fermented foods, only could produce less pectinases under general conditions. So far only a few of PGs expressed in yeast or E.coli were reported but they did not show higher activity. The cDNA of mature PGA (without signal peptide) was synthesized with specific primers from total RNA of Aspergillus oryzae by RT-PCR. PGA cDNA was ligated into pET-28a(+) expression vector, creating plasmid pET-28a(+)-pgA. The plasmid pET-28a(+)-pgA was transformed into E.coli Turner (DE3) placⅠcells to express PGA heterogeneously. For improving the efficiency of PGA expression in E.coli, the conditions for expression of the PGA in E.coli were optimized. E.coli Turner (DE3) placⅠcells with pET-28a(+)-pgA was first cultivated at 37℃，220r/min until OD_600nm reached about 0.8. Then, cultivation broth was added with 0.5 mmol/L IPTG and incubated at 15℃, 170r/min for other 24 h for induced-expression of PGA. Our data showed that the activity of recombinant expressed PGA could reach to 70u/mL medium, which is 87.5-fold of the activity of PGA produced in culture of A. oryzae and superior than known recombinant expression amount of PGA reported by other researchers.

Abstract:This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-L1 and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.

Abstract:We separated proteins of the middle silk gland through high resolution two-dimensional polyacrylamide gel electrophesis, and identified the high expressional proteins using MALDI-TOF-MS and analyzed the PMF in protein database by GPMAW（ General Protein/Mass Analysis for Windows）. The protein database was forecasted through silkworm genome database. More than 500 spots were obtained from each gel by silver stain and more than 100 protein spots were obtained from gel by Coomassie brilliant blue stain. Most of them were distributed in the area from 15kD to 90kD with pH 3.5～7. Among the 25 Coomassie brilliant blue stained spots identified by MALDI-TOF-MS, more than 60% have relatively strong signal. Comparing with the result of using Mascot, the method using PMF database of silkworm not only can identify some known proteins，but also can identify some unknown proteins that have been forecasted in silkworm genome database. Accordingly, we founded a complete set of method that fit for researching proteome of silkworm.

Abstract:The double-antibody-sandwich enzyme-linked immunoadsorbent assay (ELISA) for detection of rLysostaphin in humans had been developed and established through this study. rLysostaphin of high purity (>95%) produced in Shanghai Hi-Tech United Bio-Technological Research & Development Co., Ltd (SHUBRD) was used to produce a rabbit anti-rLysostaphin polyclonal antibody. The standard curve of rLysostaphin polyclonal antibody that was constructed showed that the lowest range of detection was found at 0.98 ng of rLysostaphin/mL, and the curve exhibited linearity preferably from 0.98 to 500 ng of rLysostaphin/mL. When three serum samples of the same batch were assayed for 6 replicates, and more 3 samples from different batches for 6 replicates, the average intra-assay and inter-assay coefficient variances (CV) were 6.4% and 6.5%, respectively. The relative recovery rate was 98.6% when quantitative standard antigens were added to the serum. The present method for detection of rLysostaphin in serum is specific, highly sensitive, highly precise, and exhibited a low CV and will be helpful in the further study of rLysostaphin pharmacokinetics and holds promise in clinical applications.

Abstract:The characteristics of gas-liquid mass transfer of three-phase system comprising air, tap water/wastewater, and hollow glass beads were studied in a laboratory-scale inverse turbulent bed reactor. The influence of operational factors and liquid property on volumetric liquid-phase mass transfer coefficient kLa was investigated under the conditions of superficial gas velocity (0.53mm·s^-1～10mm·s^-1), solid hold-up (0～0.3), and superficial liquid velocity (0～0.2mm·s^-1). The results showed that the coefficient value was 0.0456～1.414min^-1, which increased with superficial gas velocity and liquid velocity. The coefficient attained the maximum value at solid hold-up of 0.05～0.08. Compared with the coefficient value in tap water, that in synthetic wastewater and industrial wastewater is decreased by 39.0% and 50.9%, respectively. These data have provided a basis for the process analysis and mathematical simulation of inverse turbulent bed reactor.

Abstract:A quantitative structure-property relationship (QSPR) model in terms of amino acid composition and the activity of Bacillus thuringiensis insecticidal crystal proteins was established. Support vector machine (SVM) is a novel general machine-learning tool based on the structural risk minimization principle that exhibits good generalization when fault samples are few; it is especially suitable for classification, forecasting, and estimation in cases where small amounts of samples are involved such as fault diagnosis; however, some parameters of SVM are selected based on the experience of the operator, which has led to decreased efficiency of SVM in practical application. The uniform design (UD) method was applied to optimize the running parameters of SVM. It was found that the average accuracy rate approached 73％ when the penalty factor was 0.01, the epsilon 0.2, the gamma 0.05, and the range 0.5. The results indicated that UD might be used an effective method to optimize the parameters of SVM and SVM and could be used as an alternative powerful modeling tool for QSPR studies of the activity of Bacillus thuringiensis (Bt) insecticidal crystal proteins. Therefore, a novel method for predicting the insecticidal activity of Bt insecticidal crystal proteins was proposed by the authors of this study.

Abstract:Degenerate PCR primers were designed by multiple alignment of the protein sequences of known structural genes encoding the catalytic subunits of NiFe-hydrogenases obtained from Swiss-Prot Protein Sequence Database through CLUSTAL-W software and compared for conserved sequence motifs. An amplified PCR product 1 kb in size was obtained from the genomic DNA of Klebsiella pneumoniae using a set of degenerate primers, and then inverse PCR technique was used to obtain the full hydrogenase coding region. A predicted secondary structure and 3D structural model were constructed by homology modeling and docking. On the basis of these results, it was inferred that NiFe-hydrogenase from Klebsiella pneumoniae belongs to the membrane-bound H₂ evolving hydrogenase group (Ech hydrogenase group).

Abstract:Obtaining marker-free plants by high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector pNB35SVIP1 with three T-DNAs was constructed by using several mediate plasmids, in which one copy of bar gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean (Glycine max) cotyledon nodes. Through 2～3 months regeneration and selection on 3～5mg/L glufosinate containing medium, transgenic soybean plants were confirmed to be obtained at 0.83%～3.16%, and co-transformation efficiency of both gene in the same individual reached up to 86.4%, based on southern blot test. By the analysis of PCR, southern blot and northern blot combining with leaf painting of herbicide in T₁ progenies, 41 plants were confirmed to be eliminated of bar gene with the frequency of 7.6%. Among the ₁ populations tested, the loss of the alien genes happened in 22.7% lines, the silence of Tbar gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The result also suggested that the plasmid with three T-DNAs might be an ideal vector to generate maker-free genetic modified organism.

Abstract:The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3（Zhenjiang isolate）,named BmDNV-3，is a kind of bidensovirus. The most obvious characteristic in the genome of BmDNV-3 is that it has 2 sets of DNA molecular (VD1, VD2),and each of them is encapsidated respectively in the form of single-stranded liner DNA (+VD1,-VD1,+VD2,-VD2) in equal percentage. So the BmDNV-3 has 4 kinds of virions. Furthermore the sequence of BmDNV-3 is able to encode DNA polymerase itself. Some strains of silkworm revealed complete resistance to BmDNV-3, so they didn't fall sick. To investigate the difference in the process of infection and replication between the 2 virions(VD1,VD2) of this bidensovirus, and the difference of the increment in the resistant or susceptible host, the 5th instar larvae of the susceptible silkworm strain (HUABA 35 )and the resistant silkworm strain(QIUFENG d) were inoculated determinate dose of BmDNV-3 by oral ingestion. Then the midgut were collected at 9 timepoints. The silkworm cytoplasm actin A3 was used to be normalized gene, so the number of cells in collected tissue could be determined. The result shows that whatever in the susceptible silkworm strain or in the resistant one, the copies of VD1 and VD2 in the genome of BmDNV-3 collected at the different timepoint were almost at the equal level respectively, so that the VD1 and VD2 were replicated with synchronization. The process of infection in the susceptible silkworm strain was devided into 3 partitions, latent period(2～12 hours post inoculation), exponential phase (12～36 hours post inoculation)and stationary phase (36～96 hours post inoculation and there are about 2×10⁵ copies per cell). In the resistant silkworm strain, the virus were replicated at a very low level, that was from 6～10 copies 2 hours post inoculation to 150～200 copies 96 hours post inoculation (about 20 times). So we predict that the resistance in some of the silkworm strains from BmDNV-3 was a kind of chronic representation that the host carried virus without being caused flacherie.

Abstract:Two hydrogen-producing bacterial strains were newly isolated and identified as Enterobacter sp. Z-16 and Clostridium sp. C-32 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen production of strain Z-16 were achieved as: initial pH7.0, temperature 35℃, sucrose as the favorite substrate. In comparison, The optimum condition for hydrogen production of strain C-32 were obtained as: initial pH8.0, temperature 35℃, maltose as the favorite substrate . Under batch fermentative hydrogen production conditions, the maximal hydrogen conversion rate for strain Z-16 and strain C-32 were 2.68 mol H₂/mol sucrose and 2.71mol H₂/mol maltose, respectively. Using glucose as substrate, the hydrogen conversion rate of strain Z-16 and strain C-32 were 2.35 and 2.48 mol H₂/mol glucose, respectively. This research suggest a good application potential of strain Z-16 and C-32 in the future biological hydrogen production.

Abstract:The commonly used plant constitutive expression vector pBI121 was modified by insertion of two directly orientated lox sites each at one end of the selectable marker gene NPTII and by replacing the GUS gene with a sequence composed of multiple cloning sites (MCS). The resulting plant expression vector pBI121-lox-MCS is widely usable to accommodate various target genes through the MCS, and more importantly to allow the NPTII gene removed from transformed plants upon the action of the Cre recombinase. In addition, the CaMV 35S promoter located upstream of the MCS can be substituted with any other promoters to form plant vectors with expression features specified by the introduced promoters. Provided in this paper is an example that an enhanced phloem-specific promoter of the pumpkin PP2 gene (named dENP) was used to construct an NPTII-removable phloem-specific expression vector pBdENP-lox-MCS. Moreover, to facilitate screening of selectable marker-removed transgenic plants, we constructed another vector pBI121-gfp-lox-MCS in which a gfp-expression cassette is linked to the NPTII gene and the composite sequence is flanked by lox sites. Thus the selectable marker-free plants can be visually identified by loss of GFP fluorescence. The above newly created plant expression vectors can be used to develop selectable marker-removable transgenic plants for a variety of purposes.

Abstract:Previous methods used for nuclear transplantation were further investigated to develop a method that was both easy to carryout and did not require any special apparatus, such as Piezoimpact or Spindle-View. Following the puncture of zona pellucida with two holes by injection pipette that contained donor nuclei or cells, the injection pipette was pulled back to the perivitelline space while the negative pressure was increased in the holding pipette until the polar body and karyoplasm were wiped off completely. Then a reconstructed embryo was completed by the direct injection of the donor nucleus or cell without pulling out the injection pipette. 200 oocytes were manipulated using this method and it cost about 40 seconds with nucleus injection and about 30 seconds with cell injection to complete a reconstructed embryo. The success rates were 62.6% and 86.0%, respectively, and enucleation rate was about 73.3% validated by Hoechst 33342. Using this method, the nucleus was completely eliminated and another was injected using the microscope and micromanipulator. Moreover, the efficiency of nuclear transplantation and survival rate of reconstructed embryos were greatly improved. Furthermore, it is very easy to manipulate and popularize in practice.

Abstract:A reliable, low-cost, and highly efficient nonviral gene delivery system using lower molecular weight polyethylenimine (LMW-PEI) is provided. LMW-PEI was linked to an expressing plasmid with green fluorescence protein gene (gfp), the transfection activity mediated by PEIs were examined in the CM7721 cell line and the skin tissue of mouse, respectively. The cytotoxicity of PEIs, the localization and continuance time of gfp expressed in the skin tissue of mouse were also studied. Results showed that the transfection rate of gfp mediated by LMW-PEI in the CM7721 cell line was about 55%. However, with the increasing PEI molecular weight, the cytotoxicity of PEI increased, but its transfection activity decreased. The tissue transfection results showed that LMW-PEI induced a significant expression of the gfp in the cells of hair vesicle and sweat gland of mouse skin tissues following transfection of 24 h, and the expression of gfp lasted 7～9 d. When the tissue of mouse was treated with retinoic acid and nitrogenous ketone, respectively, gfp was transferred to the granule layer of mouse skin tissue. The LMW-PEI described here is a new, highly efficient vector; it would be a useful nonviral vector for gene delivery technology.

Abstract:Tissue engineering is a promising technique to repair or reconstruct the damaged cartilage in clinical use. However, chondrocyte growth is limited by the mass transport in scaffolds as diffusion is likely to be the primary mechanism. In this study, a mathematical model was developed based on oxygen diffusion and reaction to simulate chondrocyte growth. In order to accord with the fact, effective diffusion coefficients and space limitation were considered in this model and good agreement was found between experimental data and mathematical simulations. Furthermore, relationships established in the model system can be used to optimize the situation in real bioreactors and the design of three-dimensional scaffolds.

Abstract:A simple and rapid method for preparation of plasmid DNA from overnight incubation was introduced. It does not require any additional reagents; the incubation mixture containing recombinant plasmid DNA was just mixed with H₂O and phenol/chloroform/isoamyl alcohol in certain ratio. After vortexing and spinning of the mixture, the supernatant could be directly loaded onto agarose gel and analyzed using electrophoresis. The whole preparation requires only 3～5 minutes. So to quickly screen recombinant clones, this method is better compared with traditional methods.

Abstract:Environmental stresses and the continuing deterioration of arable land，along with an explosive increase in world population, pose serious threats to global agricultural production and food security. Improving the tolerance of the major crop plants to abiotic stresses has been a main goal in agriculture for a long time. As rice is considered one of the major crops, the development of new cultivars with enhanced abiotic stress-tolerance will undoubtedly have an important effect on global food production. The transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of rice cultivars. In recent years, an array of stress-related genes has already been transferred to rice to improve its resistance against abiotic stresses. Many transgenic rice plants with enhanced abiotic stress-tolerance have been obtained. This article focuses on the progress in the study of abiotic stress tolerance in transgenic rice breeding.

Abstract:Disulfide bond formation protein A, DsbA, is one of the important proteins located in E. coli periplasm, which is a foldase facilitating the folding of nascent secreted proteins, especially for those with many pairs of disulfide bonds. The crystal structure and phylogenetic analysis of DsbA and DsbA-mediated protein folding, alternatively in vivo and in vitro, are summarized. Both the extremely low pK_a of Cys^{30, about 3.5, and the destabilizing effect of the active site disulfide contribute to its strong oxidizing power. The Cys30is also considered as the most important residue closely related to its activity using site-directed mutagenesis methodology. DsbA could effectively assist proteins folding, both in vivo coexpressed with the target protein, and in vivo replenished as foldases. Moreover, DsbA also has the chaperone-like activity in the assistant refolding of genetically engineered inclusion bodies.}

Abstract:As more bioactivities of oligosaccharides have been elucidated, researches on biosynthesis of oligosaccharides have drawn more concerns in Glycobiology. A lot of enzymatic methods for the synthesis of oligosacchrides have been developed employing recombinant E. coli expressed glycosyltranferase or synthase of nucleotide-sugar. This review focuses on the recent progress in the production of oligosaccharides using bacteria especially by genetically engineered bacteria. The key point concering the oligosaccharides biosynthesis in recombinant E. coli , such as enzyme expression, NDP-sugar provision and biosynthesis pathway, was discussed.

Abstract:Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA_binding domain and four tandem repeats of the 12_aa peptide (DALDDFDLDMLG)of the herpesvirus as the activation domain,an artificial transcription factor,GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40.And then,GVP4 was cloned into expression vector pcDNA3.1/Hygro (+).Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1(+) that separately harbored EGFP cDNA and t_PA cDNA.The CHO cells were then co_transfected with GVP4 expression vector and EGFP or t_PA expression vector.The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t_PA in the supernatant.GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor.And,CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression,which resulted in 2～3_fold increase in both EGFP and t_PA expressions.These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.

Abstract:Construction of recombinant adenovirus, which contain human microdystrophin, and then transfection into mesenchymal cells( MSCs) of mdx mice were done, and genetically-corrected isogenic MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duchenne muscular dystrophy(DMD). Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease NotⅠ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by PmeⅠ, the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58×10¹² vp/mL. The recombinant adenovirus could infect MSC ofmdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin-defected MSC for stem cell transplantation to cure DMD.