Abstract:Improvement of stress tolerance to various adverse environmental conditions (such as toxic products, high temperature) of the industrial microorganisms is important for industrial applications. Ethanol produced by yeast fermentation is inhibitory to both yeast cell growth and metabolisms, and consequently is one of the key stress elements of brewer’s yeast. Research on the biochemical and molecular mechanism of the tolerance of yeast can provide basis for breeding of yeast strain with improved ethanol tolerance. In recent years, employing global gene transcriptional analysis and functional analysis, new knowledge on the biochemical and molecular mechanisms of yeast ethanol tolerance has been accumulated, and novel genes and biochemical parameters related to ethanol tolerance have been revealed. Based on these studies, the overexpression and/or disruption of the related genes have successfully resulted in the breeding of new yeast strains with improved ethanol tolerance. This paper reviewed the recent research progress on the molecular mechanism of yeast ethanol tolerance, as well as the genetic engineering manipulations to improve yeast ethanol tolerance. The studies reviewed here not only deepened our knowledge on yeast ethanol tolerance, but also provided basis for more efficient bioconversion for bio-energy production.

Abstract:FMDV 2A peptide was introduced as a linker between GP5 and M protein of porcine reproduction and respiratory syndrome virus (PRRSV) to allow automatic self-cleavage the polyproteins. This strategy simultaneously displayed the neutralizing action of GP5 protein and cell-mediated immunity of M protein. We put them into the expression cassette of adenovirus vector. The results of RT-PCR, IFA and Western blotting showed that GP5 and M protein were not only expressed correctly, but also self-cleavaged and assemble heterodimers formation. To detect the advantages of rAd-GP5-2A-M, we also constructed some other recombinant adenoviruses (rAd-GP5, rAd-M and rAd-GP5-M) as control. After inoculated subcutaneously into BALB/c mice, the four recombinant adenoviruses can induce PRRSV-specific antibodies and cell-mediated immune response, but the level of humoral and cell-mediated immune response against PRRSV induced by rAd-GP5-2A-M is the strongest among the four recombinant adenoviruses. All of these suggested that it is possible to develop one multi-gene engineering vaccine utilizing FMDV 2A peptide, and also provided a novel strategy for developing other viral disease vaccine.

Abstract:The purpose of the study is to construct recombinant goat pox virus (GPV) expressing Peste des petits ruminants virus (PPRV) H protein, and to evaluate the immunization effect. Recombinant GPV containing PPRV H gene (rGPV-PPRV-H) was selected and purified by gpt and eGFP utilizing plaque purification, and the final selected recombinant GPV was proved to be purified by PCR. Immunofluorescence and Western blotting showed that the recombinant virus could express F protein of PPRV while infecting lamb testis cells. Six goats were immunized with 2×106 PFU rGPV-PPRV-H through intradermal injection, and were immunized for the second time at 28 days with the same dose recombinant virus after first immunization. Serum was collected after immunization, and was analyzed for the neutralization antibodies. 21 days after first immunization, the neutralization antibodies of GPV were 40, 80, ≥80, ≥80, 40, ≥80 in turn, and neutralization antibodies of PPRV were 80, 80, 80, 80, 40, 40, 10 in turn; 14 days after second immunization, the neutralization antibodies of GPV were all ≥80, and the neutralization antibodies of PPRV were >80, 80, >80, 80, 80 and 40 in turn. This study established a foundation for the industrialization of the PPRV recombinant GPV vaccine.

Abstract:In order to enhance the efficiency of sheep somatic cell nuclear transfer, we used a chemically assisted enucleation with colchicine to study the effects of the concentration of colchicine, the incubation time of oocytes in colchicine and the maturation time of oocytes on the enucleation rates and the development of reconstructed embryos. The results showed that 1) there were no significant differences in the rates of cytoplast protrusion and enucleation between oocytes that were incubated in colchicine (0.4 μg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 μg/mL) for 1 h, and the rate of cytoplast protusion can be 85.4% while the rate of cytoplast enucleation is 100%. 2) There was no significant difference in oocyte enucleation between oocytes treated with medium containing 0.2 μg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 μg/mL colchicine for 0.5 h. 3) A maturation time of 18–23 h did not affect the rates of cytoplast protusion and enucleation by chemically assisted enucleation, whereas the rate of enucleation of oocytes by blind enucleation was found to decrease with a prolonged incubation time. 4) The development rates of reconstructed embryos could not be influenced by these two enucleation methods, increased from oocytes matured for 21–23 h. These results demonstrate that sheep oocytes can be enucleated fast and effectively by optimized colcholine chemically assisted enucleation, which can enhance the enucleation rate of sheep oocytes and the early development of reconstructed embryos in vitro.

Abstract:Streptococcus suis (S. suis) IgG-binding protein (SPG) was present in all S. suis strains examined. It showed binding activities with IgG from various host species. Little was known about the biological role of this protein, but it was commonly believed that it acted as virulence factor. In this study, the genes encoding SPG were amplified respectively from the total DNA of the S. suis serotype 1/2, 1, 2 and 9 with PCR and expressed in Escherichia coli BL21 by plasmid pET28a as vector. The recombinant proteins were first purified with affinity chromatography (Ni-NTA), and further purified by sephadexG-200 gel chromatography. The recombinant SPG proteins were identified to have binding activities with IgG of different host species, and for human and porcine IgG they showed better binding activities. But the SPG from different serotypes of S. suis showed no great differences in their binding activities with IgG from the same host species.

Abstract:We designed and constructed a fuse expression gene OAAT and SEA (staphylococcal enterotoxin A) on the basis of the OAAT designed and constructed which consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene. The recombinant plasmids pEA was constructed using Staphylococcal enterotoxin A (SEA) as a genetic adjuvant. Expressions of target gene from the pEA in Hela cell were verified by IFA and Western blotting. The experiment of BALB/c mice immunized with the DNA vaccines showed that pA and pEA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV, and the highest antibody titres were found in the pEA and inactivated vaccine groups compared to pA vaccinating mice. Compared with the control, the levels of IL-2, IFN-γ, IL-4, and IL-10 expression by splenic lymphocytes from mice immunized with pA and pEA were significantly increased. In addition, we found that the levels of IL-2, IFN-γand IL-4 from the mice immunized with pEA was higher than mice immunized with pA did. The results of viral challenge in guinea pigs showed the pA, pEA and inactivated vaccine provided full protection in 2/4, 2/4, 3/4, 3/4 and 4/4, 4/4 guinea pigs from challenge with FMDV O/NY00 and Asia1/YNBS/58, respectively. The results demonstrated fuse protein OAAT and SEA may be potential immunoge against FMDV, furthermore, SEA may be an effective genetic adjuvant for DNA vaccine.

Abstract:To optimize the electroporation system in Dunaliella salina (D. salina), we studied the effects of growth phase of cells, electric parameters, electroporation buffer and concentration of plasmid on transformation efficiency. The results showed that a transformation efficiency of 1.81‰ was achieved in D. salina cells at mid-log growth phase electroporated with plasmid (DCA-bar) 10 μg/mL, voltage 0.8 kV and capacitance 25 μF. However, when glycerol was added to electroporation buffer at a final concentration of 0.4 mol/L, the transformation efficiency was increased up to 2.03‰. Additionally, transformation was done with plasmids DCA-bar, NR-bar, pUΩ-bar respectively, under above optimum conditions, and similar transformation efficiencies were obtained. The findings indicate that an efficient and stable system of electroporation in D. salina has been developed, providing a powerful tool for the transgenic research of D. salina.

Abstract:Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aα-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.

Abstract:Poor stability existed in the anaphase of the high-cell-density fermentation of Saccharomyces crevisiae for S-adenosyl-L- methionine (SAM) production in 5 L fermentor. To improve the fermentation stability, we studied the addition of diammonium hydrogen phosphate, sodium glutamate and adenosine disodium triphosphate into glucose feeding solution .Study of four fed-batch cultures showed that, after 34 h fermentation, when dry cell weight reached 100 g/L, the addition of 50 g pre-L-methionine and glucose feeding with 10 g/L adenosine disodium triphosphate was optimal for SAM production. Under this condition, after 65.7 h fermentation, both the dry cell weight and the yield of SAM reached the maximum, 180 g/L and 17.1 g/L respectively.

Abstract:For expression of foreign genes in plant, plant virus vector provides many advantages, such as high expression level, short expression period and wider plant hosts. In the present study, we report the expression of thymosin alpha 1 (Tα1) in tomato fruits by potato virus X (PVX) vector. Tα1 gene fragment from plasmid pGEM-T containing Tα1 gene was cloned into plant virus vector pGR107 and the resulting pGR107-Tα1 plasmid was confirmed by digestion with Sal I and Cla I. To express the Tα1 protein, Agrobacterium tumefaciens GV3101 transformed with pGR107-Tα1 was directly injected into tomato fruits through the fruit stylar apex at different developmental stages. The ELISA results showed that Tα1 protein was expressed successfully in fruits, and the highest expression level was obtained from 2.5–3 week-old tomato fruits inoculated by bacterium at 1.0 OD600 density.

Abstract:To compare of performance of β-1,3-1,4-glucanase gene (bgl) in different expression systems, the β-1,3-1,4-glucanase gene (GenBank Accession No. EU623974) was amplified from Bacillus amyloliquefaciens BS5582 by PCR and was cloned into three vectors pEGX-4T-1, pET20b(+) and pET28a(+) to construct pEGX-4T-1-bgl, pET20b(+)-bgl and pET28a(+)-bgl recombinant plasmids. The pEGX-4T-1-bgl was transformed into three different Escherichia coli host strains. The pET20b (+)-bgl and pET28a (+)-bgl were transformed into E. coli BL21 (DE3) respectively. Recombinant β-glucanase was expressed by IPTG inducement in these recombinants. E. coli BL21 (DE3)-pET28a (+)-bgl had the highest enzyme activity. In Luria-Bertani medium, the total enzyme activity was (322.0±8.8) U/mL, which was 40.1% of original strain in optimal shaking flask condition. This recombinant’s performance was studied in Terrific Broth medium under inducement of IPTG and lactose at the same time., and the highest total enzyme activity could reach (1883.3±45.8) U/mL (818.8% of the original), which indicate that the recombinant strain has a good value in industry application.

Abstract:We studied immune modulation and antioxidant effects of wheat peptides on immunosuppressed mice. Mice were administrated with wheat peptides orally for 10 days and treated with cyclophosphamide at the 8th day. The indexes including serum hemolysin, plaque forming cells, spleen cells proliferation, liver antioxidant enzymes activties, MDA, scavenging serum 2,2-Diphenyl-1-picrylhydrazyl and ·OH and macrophage phagocytic ability in vitro were measured to assess the immune functions and antioxidation abilities. In vivo study shows that cyclophosphamide significantly decreases serum hemolysin (HC50) and phagocytic function of macrophages. Simultaneously, liver superoxide dismutase, catalase activity and total oxidation capacity were decreased and malondialdehyde was increased. Wheat peptides could recover HC50 and spleen cell proliferation when orally administrated. Furthermore, they could also enhance serum 2,2-Diphenyl-1-picrylhydrazyl and ·OH scavenging. In conclusion, wheat peptides can help body resist the stress related disorders in immune and antioxidant system.

Abstract:We developed single base extension-tags (SBE-tags) microarray to detect eight common food-borne pathogens, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazaki, Shigella, Escherichia coli O157:H7 and Campylobacter jejuni. With specific PCR primers identified and integrated for eight food-borne pathogens, target sequences were amplified and purified as template DNA of single base extension-tags reaction. The products were hybridized to microarrays and scanned for fluorescence intensity. The experiment showed a specific and simultaneous detection of eight food-borne pathogens. The system limits is 0.1 pg for a genomic DNA and 5×102 CFU/mL for Salmonella typhimurium cultures. The single base extension-tags assay can be used to detect food-borne pathogens rapidly and accurately with a high sensitivity, and provide an efficient way for diagnosis and control of disease caused by food-borne pathogens.

Abstract:LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Further Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.

Abstract:Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Bat-PA (H), the full-length form of Vampire Bat Salivary Plasminogen Activator (DSPAa1), is homologous and similar efficacy to tissue-type plasminogen activator (t-PA). The strict fibrin dependence of activity is a characteristic which could be desirable in the fibrinolytic therapy. It is a unique fibrinolytic enzyme that does not promote neurodegeneration. In this study, according to the reported gene sequence (GenBank Accession No. J05082) of Vampire bat (D. rotundus) plasminogen activator. It was the first time to synthesize the full sequence of DSPAa1 in vitro and clone it into the expression vector pPIC9K, the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 strain. Secreted expression of recombinant DSPAa1 was attained by methanol induction and its molecular mass is 47 kD. To get recombinant GS115 with high amount of protein, hundreds of His+ transformants had been screened to isolate clones resistant to high levels G418 (2–4 mg/mL), the selected clones mini-expressed in Pichia pastoris, and test their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAa1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAa1 derived often from mammalian cells: Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, COS cells, which might be produced at high cost. In Pichia pastoris, it is expected to higher yield and lower cost, thus it might be able to serve as new thrombolytic candidate.

Abstract:The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.

Abstract:To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.

Abstract:miR-122 is the most abundant miRNA in adult human liver. To study the functions of miR-122 in liver disease, we amplified the precursor of human miR-122 gene by polymerase chain reaction (PCR) from the HepG2 genomic DNA, and then constructed miR-122 expression vector pLMP-miR-122. pLMP-miR-122 could overexpress mature miR-122 when human normal liver cells L-O2 and the hepatoma cells HepG2 were transfected with it. When HepG2 cells were co-transfected with HBV1.3 and pLMP-miR-122, we found that miR-122 could down-regulate the expression of HBs and HBe antigen. These results showed that the human liver specific miR-122 expression vector was constructed successfully, and it could regulate the replication and the expression of HBV genes. The plasmid pLMP-miR-122 will facilitate further studies of the functions of miR-122 in the development of liver virus infection diseases and HCC.

Abstract:Human-like collagen (HLC) was cross-linked with hyaluronic acid by genipin in different ratio. The concentrations of hyaluronic acid in the mixture were 0, 0.01%, 0.05% and 0.1%. The blood vessel tubular grafts were then fabricated by freeze-drying. Microstructure, element composite, mechanical properties, cytotoxicity grade, and biocompatibility of different vascular scaffold groups were studied by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), tensile test, burst pressure experiment, cytotoxicity experiment, endothelial cells planted in blood vessel scaffolds and hypodermic embedding of mice. The results showed that HLC-HA (0.05%) tubular scaffold exhibited interconnected well-distributed and porous structure and porosity of 94.38%; achieved the desirable mechanical property with stress of (1000.8±7.9) kPa and burst pressure of (1058.6±8.2) kPa, hypocytotoxicity, favourable cytocompatibility, hisocompatibility and disposition of degradation.

Abstract:Adult pluripotent stem cells, such as mesenchymal stem cells derived from bone marrow and adipose tissue are capable of multilineage differentiation. Although autologous stem cell transplantation is an effective alternative to organ transplantation, the loss of cell viability and differentiation confinement of implanted cells has largely impaired the therapeutic efficacy. To produce biomaterial-free liver construct to integrate into living tissue, we isolated adipose mesenchymal stem cells and subjected them to a delicate culture configuration to mediate the hepatocyte differentiation. The differentiated hepatocyte-like cells were then inoculated onto poly (N-isopropylacrylamide) (PNIPAAm) grafted cell culture dish. By lowering the culture temperature to 20oC, cells detached from the dish surface into a complete cell sheet. Hematoxylin and eosin staining and immunohistochemistry results showed that cell sheet was composed of 2–3 layers of cells and extracellular matrix was maintained intact. As compared with traditional cell harvest using trypsin digestion, cell sheet fabrication causes no damage to cell membrane and extracellular matrix. Hence, cell sheets would form a better interaction with tissues in situ, and a higher cell viability and therapeutic efficiency would be expected.

Abstract:Polymerase chain reaction (PCR) is a simple, quick and highly sensitive method. However the accuracy of the conventional PCR assay was often affected by false positives and false negatives. In this study, a protocol competitive PCR was used to reduce the false results in screening for selectable marker-free (SMF) Xa21 transgenic rice plants. The competitive template of Xa21 was the endogenous Xa21 homologous sequence located on chromosome 11. The competitive template of the selectable marker gene, hygromycin phosphotransferase (hpt), was an additive DNA extracted from hpt transgenic Nipponbare (Oryza sativa L). Through competitive PCR analysis of transgenic T1 plants produced by double right border binary vector, false positive or false negative samples were effectively diminished, and genuine SMF Xa21 transgenic plants were obviously obtained. Comparing with the conventional non-competitive PCR, competitive PCR increased the accuracy for selecting SMF Xa21 transgenic plants. The results of bacterial blight (BB) resistance tests and hygromycin B resistance assay of SMF Xa21 transgenic plants testified the reliability of this method.

Abstract:Polystyrene microspheres (PS) were successfully prepared by suspension polymerization processes. Chloroacetylated polystyrene has been prepared by Friedel-Crafts acetylation of PS with chloroacetyl chloride. In this report, carcinogenic compound (chloromethylether etc.) was avoided. The effects of solvent, catalyst, acylating agent and reaction time were studied. Novel adsorption resins were obtained by synthesis of chloroacetylated polystyrene with amine. The influences of solvent, amine reagent and reaction time on ion exchange capacity were investigated. Under the optimized reaction condition, the ion exchange capacity of the prepared resins was 4.1587 mmol/g. The maximum amount of adsorbed bilirubin was 30.85 mg/g, the adsorption percentage was 80%.

Abstract:In order to improve the stationary phase retention of polar solvent systems and aqueous two-phase systems (ATPSs), we designed a multiple spiral disk assembly for type-J high-speed counter-current chromatography (HSCCC). The stationary phase retention was studied under different elution modes by using two solvent systems that contained 1-butanol-acetic acid-water (4:1:5, V/V/V) and polyethylene glycol (PEG) 1000-K2HPO4-water (12.5:12.5:75, W/W/W). The best retention was obtained in L-I-T, U-O-H, L-I-H three modes by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a relatively high flow rate. Meanwhile, the relationship between retention percentage of the stationary phase retention (Sf) and various parameters such as flow-rate (F), rotation speed (w) and column temperature (T) was also studied. Sf increased with the increase of w and decreased with the increase of F. Regression analysis showed a linear relationship between Sf and F1/2/w. The influence of T on Sf was not obvious between 20°C and 40°C, lower temperature than 20°C was not suitable for viscous ATPSs. Acceptable resolutions were achieved when it was applied for the separation of dipeptides including Leu-Tyr and Val-Tyr by using 1-butanol-acetic acid-water (4:1:5, V/V/V) solvent system. The proteins including cytochrome C and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins were well separated by 12.5% PEG1000-12.5% K2HPO4-75% water (pH 9.0) and 16% PEG 1000-12.5% K2HPO4-71.5% water (pH 8.0) system.

Abstract:A recombinant strain Escherichia coli DH5a(pMD19-glnA) including Bacillus subtilis glnA gene was constructed. Capillary electrophoresis and nuclear magnetic resonance were used to determine qualitatively the product of transformation by recombinant strain, and the relative level of mRNA expression of glnA was also determined by fluorescence quantitative RT-PCR. Subsequently, SDS-PAGE (polyacrylamide gel electrophoresis) was used to analysis the relative level of protein. Surprisingly, there was no increase of glutamine production in this recombinant strain, but an obvious increase in the GABA (g-aminobutyric acid ) production. It was showed in the experiment that protein expression of the glutamine synthetase did not increase, although glnA gene can be transcribed normally in this recombined strain. The phenomenon of exogenous glnA gene interfering metabolism of Escherichia coli was worthy of further study.