Abstract:Resveratrol is a naturally occurring stilbene, a kind of polyphenolic compounds, found in a limited number of plant species such as grape, peanut, and pine. It has been considered as a phytoalexin in plants, and many studies have also shown its health benefits such as antioxidant activities, cancer prevention, blood thinning, and life span extension. This paper reviews the characteristics of resveratrol in aspects of synthesis, extraction, purification, and determination. In particular, the new outcomes of physiology function and the transgenic approaches have been presented. The challenges and chances for genetic engineering and heath-related industries were also discussed.

Abstract:Phytohormone auxins play important roles in plant growth and development. The primary auxin-response genes can be classified into three major groups: Aux/IAAs, SAURs and GH3s. Significant progress has been made in understanding these gene families by approaches of the functional genomics, molecular genetics and molecular biology. In this review, we focused on the structures, functions and models of the expressional regulation of plant GH3 genes. The interactions in the signal transduction pathways between auxins and other signals mediated by the GH3 genes, the relationship between the GH3 genes and the stress adaptation responses of plants are emphasized.

Abstract:Cold acclimation can improve freezing tolerance. Here cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to isolate differentially expressed cDNAs and a Pp-LIM only A cDNA was isolated and identified in the cold acclimation of Physcomitrella patens. Real-time RT-PCR indicated it is obviously up-regulated at 6 h, 12 h, 24 h, 48 h and 72 h after cold acclimation. After comparing the cDNA with the gene sequence, seven introns and eight exons were identified in the cDNA. The cDNA putatively encodes a protein of 345 amino acid residues and only contains one LIM domain which has highly similarity with the PDZ/LIM domains of the protein family in animals. We proposed that Pp-LIM only A be a new gene coding for the LIM-domain containing protein and enhance stability of cell membrane via their effects on cytoskeleton during cold acclimation in P. patens.

Abstract:To prolong serum half-life of human Erythropoietin for better efficacy, a new form of recombinant human erythropoietin (rhEpo-L-Fc) was generated by fusion of a full length human erythropoietin gene and the Fc fragment of human IgG1 with flexible linker sequence. The fusion gene rhEPO-L-Fc was constructed by PCR, then inserted into expression vector pOptiVEC?-TOPO?, and expressed in Chinese Hamster Ovary cells deficient in the DHFR enzyme(CHO-dhfr-). The chimeric protein was purified by Protein A affinity chromatography, showed expected molecular weight and demonstrated a similar bioactivity compared to that of the native recombinant human erythropoietin (rhEPO) in an EPO-dependent cell-based assay. In vivo pharmacokinetic studies showed that the rhEPO-L-Fc had an elimination half-life of 27 h. In vivo efficacy studies showed that a single dose administration of rhEPO-L-Fc in rats increased the reticulocyte number in the peripheral blood significantly. These results demonstrated that the new engineered rhEPO-L-Fc may become alternative therapeutic approach to extend the half-time of rhEPO to treat anemia.

Abstract:Fas-associated death domain (FADD) is a signal connection protein in Fas/FasL apoptotic path which might play a key role on apoptosis by transferring apoptotic signal. To reveal the intracellular signal transduction molecules involved in the procedure of follicular development in bovine ovary, we cloned FADD gene in bovine ovary tissue with RT-PCR, deleted the termination codon in its cDNA and directionally cloned the amplified FADD gene into eukaryotic expression vector pAcGFP-Nl including AcGFP, successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme Bgl II/EcoR I and sequencing, transfected pAcGFP-bFADD into CHO-K1 cell mediated by Lipofectamine 2000, observed the expression of AcGFP and detected the transcription and expression of FADD by RT-PCR and Western blotting. The results showed that the cattle FADD was successfully cloned, the pAcGFP-bFADD fusion protein recombinant plasmid was successfuly constructed by introducing Bgl II, EcoR I cloning site at two ends of FADD open reading frame and inserting a Kozak sequence before start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 654 bp transcription was amplified by RT-PCR, and 51.4 kD target protein was detected by Western blotting. Construction of pAcGFP-bFADD recombinant plasmid should be helpful for further understanding the mechanism of regulation of FADD on bovine oocytes formation and development.

Abstract:The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or △LNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and △LNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of △LNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated △LNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.

Abstract:We established a cell based high throughput screening model by calcium assay on fluorometric imaging plate reader for finding modulators of TRPV3．The TRPV3 expression vector was transfected into HEK-293 and table cell line expressing TRPV3 was selected with antibiotics. Upon TRPV3 specific modulators stimulated, pharmacological characteristics of TRPV3 over expression cell line were detected by calcium assay on fluorometric imaging plate reader. Assay conditions were optimized and stability of the model was observed. The reliability and accuracy of application to 96 and 384 well format high throughput screening were also evaluated. A stable HEK-293 cell line highly expressing TRPV3 was established. TRPV3 specific modulators could modulate calcium signal through TRPV3 in dose dependent manner. Optimized screening condition was established by assay development. This model is stable and sensitive, and meets the requirement of high throughput screening by Z'factor validation and Spiking test. This cell model can be applied to screening TRPV3 modulators by calcium assay.

Abstract:The M2 ion channel protein is an important target against influenza A virus. In this study, H5N1 influenza A virus M2 ion channel (H5M2) gene was cloned into pcDNA4 vector. The HEK293 stable cell line expressing H5M2 was successfully established. The expression of H5M2 ion channel protein was induced only by tetracycline and confirmed by imuunofluorescence and Western blot. The ion channel activity of H5M2 was confirmed by whole cell patch-clamp recording. Fifty mmol per liter amantadine blocked the H5M2 channel conductance completely in HEK293 cells. This stable cell line may provide a model for screening inhibitors of M2 ion channel.

Abstract:Collagen and chitosan are well natrual polymers to be used as extracellular matrix on tissue engineering because of their biocompatibility, certain mechanical strength and biodegradability. But there are some disadvantages when they are used to construct extracellular matrix respectively. This experiment utilized their complementary performances to prepare a composite extracellular matrix of artificial tendon tissue that had adequacy mechanics strength and good biocompatibility, cell affinity, biodegradability. Collagen and chitosan were covalently crosslinked using EDC and NHS to obtain a porous scaffold material that the porous was oriented under an external force. Then RGD peptide was covalently attached to scaffold material surface to improve its affinity with cells. The microstructure of scaffold material was observed under microscope and scanning electron microscope. Simultaneously, the physical performance, hydrophilicity, ecto-degradation rate and cell compatibility of scaffold material were measured in the experiments. The results showed that this scaffold material was soft and stretchy. Its tensile strength was 15.0 MPa, corresponding shape extension was 7.33%, and its porosity was 79.4%. Its water absorption rate and water retention rate were 772% and 206% respectively. Its degradation rate in RPM1640 culture mediun with 10% fetal bovine serum and in human serum were 4.13% and 37.2% respectively after three weeks. These degradation rates are suitable for the rehab course of injured tendon. Moreover the degradation rates can be controlled by adjusting technological conditions and degree of cross linking. Significantly higher affinity with 3T3-L1 cell was detected on the scaffold material modified by RGD peptide. The various physical performances of this complex scaffold material are appropriate for constructing extracellular matrix of artificial tendon tissue or artificial skin. Moreover, it could be used as soft tissue slurry for plastic surgery.

Abstract:NS1 is a non-structural protein of the influenza A virus, which could only be expressed when cells are infected. The effect of NS1 protein on host cell is still not clear. To understand the role of NS1 protein in cell infection, recombinant plasmid pCMV-myc-NS1 was constructed, and then transfected into A549 cells. Two-dimensional electrophoresis was employed to analyze proteins regulated by NS1 that could reflect the interaction between influenza virus and host cells at the protein level. The influence of NS1 on cell proliferation and cell cycle was also studied. The result showed that not only could NS1 remarkably affect metabolism, but it could also slow down cell proliferation through blocking cell cycle.

Abstract:We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.

Abstract:A library with potential to produce six amino acids cyclic peptides was prepared using pET-28a as the starting plasmid. pVmut was used to amplify the IntC-dnaB-N-IntN fragment that was inserted into pET28a to give pEV. On pEV, DnaB split intein was expressed under the strong T7 promoter. Analyses of Escherichia coli transformed with pEV showed that DnaB split intein was produced in large quantity and the fusion protein self-spliced efficiently to produce cyclized DnaB-N. A synthesized 115 bp fragment mixture encoding 5 random amino acids was inserted into pEV to generate pEV-IS. The ligation mixture was transformed into E. coli. A library of 103 clones was obtained, 20 randomly picked clones were sequenced. All of them contain different sequences. Nine clones were chosen for further analysis. Split-intein-ISs were expressed in large quantity, and 90% of them self-spliced under 16°C in 20 hours. After induction at 30°C for 3 hours, the expressed DnaB split intein was purified using His-column, and then a molecular weight of target cyclic peptide was detected by MALDI-TOF-MS.

Abstract:A unique one-step ethanol fermentation process was developed with the inulinase-producing strain Kluyveromyces marxianus YX01. Firstly, the impact of temperature on ethanol fermentation was investigated through flask fermentation, and the temperature of 35oC was observed to be the optimum to coordinate inulinase production, inulin saccharification and ethanol fermentation. And then, the impact of aeration and substrate concentration was studied through batch fermentation in the 2.5 L fermentor, and the experimental data indicated that the average ethanol fermentation time was decreased at the aeration rates of 50 mL/min and 100 mL/min, but higher ethanol yield was obtained under non-aeration conditions with more substrate directed to ethanol production. The ethanol concentration of 92.2 g/L was achieved with the substrate containing 235 g/L inulin, and the ethanol yield was calculated to be 0.436, equivalent to 85.5% of its theoretical value. Finally, Jerusalem artichoke grown in salina and irrigated with seawater was fermented without sterilization treatment, 84.0 g/L ethanol was obtained with the substrate containing 280 g/L dry Jerusalem artichoke meal, and the ethanol yield was calculated to be 0.405, indicating the Jerusalem artichoke could be an alternative feedstock for grain-based fuel ethanol production.

Abstract:Astaxanthin is a useful pigmentation source in fish aquaculture. It has strong antioxidative activity and therefore has potential application in delaying aging and degenerative diseases in human and animals. In recent years, there is a growing demand for astaxanthin. The red yeast Xanthophyllomyces dendrorhous (called Phaffia rhodozyma before) is one of the most promising microorganisms for the commercial production of astaxanthin. During fermentation, X. dendrorhous shows the Crabtree effect. Higher glucose concentration will cause significant reductions in biomass and astaxanthin production. Therefore, fed-batch processes are particularly useful. In this paper, effects of glucose-feeding strategies on astaxanthin production by X. dendrorhous were studied. Based on the substrate inhibition model, an optimized two-stage feeding strategy for astaxanthin production of high-cell-density fermentation was proposed. Glucose concentration was first controlled at about 25 g/L during the lag phase and the early exponential phase. In such case, biomass could reach its maximum value in relatively short time. Then the glucose concentration was controlled at about 5 g/L in the later exponential phase and stationary phase. The synthesis of astaxanthin could be effectively prolonged. The results showed that the optimized two-stage feeding strategy was the best among all the feeding strategies, and could obtain the highest biomass (23.8 g/L) and astaxanthin production (29.05 mg/L), which was a significant increase (52.8% and 109% respectively) compared with a batch process.

Abstract:The products concentrations in traditional acetone-butanol (AB) fermentation are too low that large amount of energy has to be consumed in the distillation and product recovery process. Aiming at direct utilization of the fermentation products, in this study, optimization of property-improved biodiesel manufacturing process coupled with AB extractive fermentation was conducted, under the condition of using the biodiesel originated from waste cooking oil as the extractant and high concentrated corn flour medium. The effect of biodiesel/broth volume ratio, waste supernatant recycle ratio, and electronic carrier addition on the major process performance index was carefully investigated. Under the optimized condition, the biodiesel quality was improved with the cetane value increased from 51.4 to 54.4; “actual butanol yield” reached to a level of 18%, and waste supernatant recycle ratio exceeded 50%. In this way, elimination of energy-consuming product recovery process and realization of “energy-saving & waste minimization” industrial production target advocated by the state government, could be potentially expected.

Abstract:Vasostatin, a 180-amino acid fragment from the N-terminal domain of calreticulin, is a potent endogenous angiogenesis inhibitor, which can inhibit the growth of many kinds of experimental tumor. But a recent report that vasostatin can enhance the malignant behavior of neuroendocrine tumor reminds us to be cautious to develop it as an anti-tumor medicine. VAS cDNA was cloned into pAAV-2 expression vector; recombinant virus rAAV-VAS was generated by a three plasmids, helper free packaging method. MS1 mouse pancreatic endothelial cell and human colon tumor HCT-116 cell were infected with rAAV-VAS. Transgene expression was analyzed by Western blotting analysis; cell proliferation was determined by MTT assay. The therapeutic potential of rAAV-VAS was evaluated in subcutaneous HCT-116 xenograft mouse model. rAAV-VAS inhibited the proliferation of MS1 but not HCT-116 cell. HCT-116 cell infected with rAAV-VAS secreted VAS protein into the supernatant effectively. The intratumoral delivery of rAAV-VAS inhibited the xenograft growth and microvessel density in tumors significantly. Our results show the effectiveness of rAAV-VAS as an angiogenesis inhibitor in suppressing tumor growth, validating the application of rAAV-VAS gene therapy in treatment against colon cancer.

Abstract:To improve thrombolytic effect, a fusion protein SFH composed of staphylokinase (SAK) and hirudin (HV) with blood coagulation factor Xa (FXa) recognition peptide as a linker, was designed. SFH showed improved thrombolytic effect and low bleeding in vivo. Two thrombus-targeting mechanisms might account for the above features of SFH. This study was designed to study the two thrombus-targeting mechanisms of SFH. ELISA and immunohistochemistry assay were used to study the improved thrombus selectivity of SFH and the results showed that SFH, compared with SAK, displayed higher affinity for thrombin and thrombin-rich thrombus. To verify the thrombus-targeting release of anticoagulant activity of SFH, FH-a derivative of HV with only FXa recognition sequence at N terminus of HV was designed and used in animal tests. In inferior vena cava thrombosis model, FH showed equal antithrombotic effect as HV, indicating that HV could be successfully released from FH by FXa cleavage in vivo. More importantly, no prolongation of plasma TT, APTT and PT were found in FH group, but significant prolongations were discovered in HV group. This revealed that the anticoagulant activity of FH was released in thrombus-targeting way and limited in the vicinity of the thrombus, and this could be extrapolated to SFH. In conclusion, the high thrombus affinity and thrombus-targeting release of anticoagulant activity of SFH assigned low bleeding risk to SFH.

Abstract:Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessel. We prepared recombinant extracellular domain 3 (KDR3) of human vascular endothelial growth factor receptor-2 in Escherichia coli and studied its specific binding activity with its ligand. The target DNA was synthesized by overlapping PCR, ligated with expression vector p-ET32a and transformed into E. coli Rosetta (DE3). The soluble fusion protein Trx-KDR3 was expressed in cytoplasm, which was up to 20% of total soluble protein in cytoplasm after having been induced by 1 mM IPTG for 5 h at 30℃. It was characterized to be target protein by Western blotting. The product was purified by CM cation exchange resin and immobilized metal affinity chromatography(IMAC). Its VEGF-binding activity was determined by ELISA assay and its influence on the propagation of HUVEC induced by VEGF. The protein product showed high ligand binding activity in the ELISA and HUVEC propagation study compared to the control. Therefore, ligand binding active, soluble recombinant extracellular domain 3 of VEGFR-2 KDR was successfully expressed and purified in E. coli, which would be applied to anti-angiogenesis anti-tumor therapy and anti-KDR antibody development.

Abstract:Lipases are widely used enzymes in biotechnology. Although they catalyze the same reaction, their sequences vary. Therefore, it is highly desired to develop a fast and reliable method to identify the types of lipases according to their sequences, or even just to confirm whether they are lipases or not. By proposing two scales based pseudo amino acid composition approaches to extract the features of the sequences, a powerful predictor based on k-nearest neighbor was introduced to address the problems. The overall success rates thus obtained by the 10-fold cross-validation test were shown as below: for predicting lipases and nonlipase, the success rates were 92.8%, 91.4% and 91.3%, respectively. For lipase types, the success rates were 92.3%, 90.3% and 89.7%, respectively. Among them, the Z scales based pseudo amino acid composition was the best, T scales was the second. They outperformed significantly than 6 other frequently used sequence feature extraction methods. The high success rates yielded for such a stringent dataset indicate predicting the types of lipases is feasible and the different scales pseudo amino acid composition might be a useful tool for extracting the features of protein sequences, or at lease can play a complementary role to many of the other existing approaches.

Abstract:hUBEW, a newly identified class I ubiquitin conjugating enzyme, probably plays an important role in tumorigenesis and DNA repair processes. RNA interference (RNAi) is a process in cells to degrade specific homologous mRNA by forming duplex RNA and has been developed into a powerful tool to study gene functions. In this study, the H1-U6 dual promoter RNAi plasmid was constructed and the target sequence for hUbe2w could be transcribed from both strands and form a double stranded RNA with two 5'Uridine overhangs, which closely resembles endogenous functional siRNA. The hUbe2w cDNA was amplified from reverse transcription of the 293FT total RNA by RT-PCR, and then cloned into the pGL3-Control, pCMV-myc and pDsRed-express-C1 plasmids respectively, which were selected as report vectors to detect the RNAi effects. The plasmids were co-transfected into HEK293FT cells, and then the luciferase activity and hUBE2W protein expression were measured respectively. The Resulted reduction of mRNA and protein level demonstrate that the targets of 125 and 259 could significantly inhibit the hUbe2w expression.

Abstract:In order to identify rat ovarian germ cells, we expressed and purified rat RVLG protein in Escherichia coli cells and prepared a mouse anti-rat RVLG polyclonal antibody. The rat RVLG cDNA was obtained from rat testicle tissue by RT-PCR and was cloned into the vector pMD19-T. Sequence analysis proves that the cloned RVLG cDNA fragment was 60 bp longer than that released in the GenBank (NM_001077647), resulting from an alternative splicing of the RVLG pre-mRNA. The RVLG cDNA was double digested with the restriction endonucleases BamH I and EcoR I, and then was extracted from gel and inserted into the prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmid pGEX-RVLG was verified for successful construction and then was transformed into Escherichia coli BL21(DE3) for induction to express the GST-RVLG fusion protein by IPTG. The GST-RVLG fusion protein was expressed in Escherichia coli BL21 (DE3) at a high level which accounts for more than 10% of the total bacterial cellular protein. The purified RVLG protein was used as an antigen to immunize KM mouse for the production of polyclonal antibody in ascetic fluid followed by celiacly injecting the mouse with S180 cells. The mouse anti-rat RVLG antibody was analyzed by ELISA, Western blotting and immunohistochemistry for its specificity and titer. The antibody could recognize RVLG protein specifically and its titer was about 1:20 000. These results confirm that the mouse anti-rat RVLG polyclonal antibody with high affinity and specificity has been prepared successfully, and lay a foundation for our ongoing research on the specific expression of RVLG in rat ovary.

Abstract:Promoter-trap strategy for enriching targeted colonies has been usually used to elevate the gene targeting efficiency in somatic cells. Knocking out Prnp in animals by gene targeting can render them resistant to Prion diseases. We constructed a bovine Prnp promoter-less targeting vector BoPrPneo, then transfected the linearized vector into the bovine fetal fibroblasts BFF through electroporation. After selecting in cell culture medium with 250 μg/mL G418, we obtained 99 drug-resistant cell colonies, 4 of them were positive for targeted events after PCR screening, and the targeted colonies were further confirmed by sequencing and Southern blotting. This suggests that one allele of Prnp has been successfully knocked out in bovine fetal fibroblasts. This research supplies a simple, safe and effective method to targeting bovine Prnp.

Abstract:Bioleaching of Cu and Fe in low-grade chalcopyrite using Penicillium janthinellum strian GXCR was studied. As a result, shaking bioleaching was more efficient than submerged bioleaching; Cu bioleaching was much better than Fe bioleaching; under conditions of optimum carbon source (10% sucrose, W/V), optimum nitrogen source (1.5% NaNO3, W/V), shaking bioleaching and the optimum combination of conditions (initial pH 6.0 in leaching media, 5 % (W/V) 200–mesh ore and initial inocula of 3.0×105 conidia/mL), Cu bioleaching efficiency reached 87.31% (W/W). One of the most important factors affecting Cu bioleaching in shaking bioleaching was the initial pH in leaching media (F> F0.05). The major organic acids for Cu and Fe bioleaching were citric and oxalic acids, respectively. Low bioleaching efficiency by submerged bioleaching was due to low production of citric and oxalic acids. The mechanisms employed by the GXCR in Cu bioleaching included biochemical functions of citric and oxalic acids as well as ore crack caused by mechanical power generated from mycelial growth.