Abstract:Recombinant adeno-associated viral vectors (rAAV) have been widely used as gene therapy vectors in clinical trials. Here, we reviewed the genomic structures and replication mechanisms of wt-AAV. Then, the assembly of capsid and the encapsidation of genomic DNA, two major events during AAV pakaging, was discussed in detail. Although the overall pattern of virus assembly and encapsidation is known, the molecular mechanisms and the structure-function relationship involved in these processes are not well understood. Further elucidatation of these processes may improve the production technology of rAAV and develop gene drug based on rAAV.

Abstract:DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.

Abstract:Cation transporters play important roles in modulating the concentration of intracellular metal ions. The vacuole is an important storage organelle for many ions. Cation (Ca2+)/H+ antiporters (CAXs) located at vacuolar membrane are mainly involved in the Ca2+ flux into the vacuole, and appear to be capable of transporting various divalent cations to some degree. Several CAX genes have been isolated and characterized from various plants in recent years. Four domains of plant CAXs have been identified: NRR regulates Ca2+ transport by a mechanism of N-terminal autoinhibition; Ca domain and C domain confer Ca2+ and Mn2+ specificity among CAX transporters, respectively; D domain plays a part in the regulation of cytosolic pH. AtCAXs identified in Arabidopsis thaliana are involved in the growth, development and stress adaption of plant. AtCAX3 is the mainly Ca2+/H+ transporter in response to salt stress; AtCAX2 and AtCAX4 participate in transportation and detoxicification of heavy metal ions (Cd2+, Zn2+, and Mn2+) in cells under heavy metal stress, and impact root/shoot Cd partitioning in plant. These suggest that CAX genes may be useful for nutritional enhancement of plants, and for increasing phytoremediation potential. Here, the classification, structure and function of CAXs in plants are reviewed.

Abstract:To screen the interactive proteins with IBDV Gt VP2 protein from cDNA library of B Lymphoid cells of the bursa of fabricius. The expression cDNA library plasmids was transformed to the yeast competent cells, which have the bait plasmid-Gt VP2. After testing for growth in synthetic complete medium lacking histidine and uracil and for production of b-galactosidase (X-gal), we obtained 16 positive clones. We searched the gene sequences of positive clones in the NCBI website. The blast results showed that five positive clones were the gallus sequences. They were Gallus gallus breed mitochondrial DNA, O_GlcNAc transferase, Tumor protein p53 binding protein, Stathmin and Chondroitin sulfate GalNAcT-2, respectively. This study is helpful for the further identifying the receptors of IBDV in B Lymphoid cells of the bursa of fabricius.

Abstract:In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface Isdb, we amplified isdb gene from S. aureus Wood46 strain. The isdb gene was subsequently inserted into pET32a(+) vector and the recombinant plasmid was transformed into E. coli strain BL21. The recombinant Isdb was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level was measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus strains Wood46 and HLJ23-1. These results showed that isdb gene sequences were highly conserved, and the recombinant Isdb was successfully expressed. The antibody titer in the immunized groups was increased significantly (P<0.05) compared with the control, the protective rate of Isdb protein inducted by challenge with the two S. aureus stains Wood46 and HLJ23-1 was 62.5% and 75%, respectively. These results showed that the Isdb protein had high immunogenicity and immunoprotective capacity.

Abstract:Construction and ethanol production effects of SNF4 gene knockout in Saccharomyces cerevisiae were described in this paper. For knockout of SNF4 gene in S. cerevisiae YS2, a PCR-amplified disruption cassette was used, encoding the short flanking homologous regions to the SNF4 gene and Kanr as selectable marker. The SNF4 gene disruption cassette was transformed into S. cerevisiae YS2 through LiAc/SS Carrier DNA/PEG. The positive transformants were grown on G418 plates and verified by PCR. The Kanr marker was rescued by transforming plasmid pSH65 into positive transformants and inducing expression of Cre recombinase in galactose-containing medium. Lastly, the YS2-△SNF4 strain, in which SNF4 allele gene were completely knocked out, was obtained by repeating the same procedure. The result of anaerobic fermentation showed that ethanol production of the SNF4 gene knockout strain had increased by 7.57 percent as compared with the original strain YS2. The experiment indicated ethanol production could be improved significantly with the approach of SNF4 gene knockout by Cre-LoxP system.

Abstract:Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate is a potential intermediate for the synthesis of HMG-CoA reductase inhibitor (atorvastatin) that can lower the cholesterol level in human blood. In this study, in order to synthesize ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate by bioreduction, the yeast strains in our lab were screened. Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate was found to be produced efficiently from ethyl 5-(1,3-dioxoisoindolin-2-yl)-3-oxopentanoate by Pichia pastoris X-33. The effects of initial substrate concentration, reaction time, co-substrate, amount of yeast cells, pH, as well as the temperature on the yield and enantiomeric excesses (e.e. value) of product were examined in mono-phase system. The optimal reaction conditions are as fallows: substrate concentration 7 g/L, cell concentration 120 g/L, glucose concentration 120 g/L, pH 6.5, temperature 35 °C, reaction time 12 h, and the yield 93.12% with the high e.e. value of 98.55%.

Abstract:We have developed a rapid and high throughput lipase-ANS (8-Anilino-1-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal’s increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 °C to 65 °C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement.

Abstract:In order to learn the enzymatic hydrolysis characteristics of steam-explosion pretreated corn straw by cellulase, the effects of substrate concentration, cellulase concentration and temperature were determined. The kinetics of the hydrolysis reaction could be described with the Michealis-Menten equation, and the hydrolysis reaction obeyed the classical first-order reaction rate in the first three hours. In the condition of 45 °C and pH 5.0 and the stirring rate 120 r/min, the Michealis constant (Km) and maximum rate (Vm) for 1.2 FPU/mL of cellulase were 11.71 g/L and 1.5 g/(L·h). The kinetic model, including the parameters such as substrate concentration, enzymatic concentration and temperature, was suit for the hydrolysis reaction under the temperature range from 30 °C?50 °C.

Abstract:A comparative study of batch and repeated batch process was carried out for astaxanthin fermentation of Phaffia rhodozyma to develop a more economical method for astaxanthin industrial production. In shaking flask fermentation, the change of biomass and astaxanthin production was studied to compare the five-day cycle with four-day cycle of repeated batch culture of P. rhodozyma. Astaxanthin production increased at first and then decreased subsequently in seven cycles, yet the yield of astaxanthin in the next six cycles remains higher than that of the first cycle. Comparing the average production of astaxanthin in the seven cycles, four-day cycle performed even better than five-day cycle. Subsequently, a repeated fed-batch process was used in a 5-l bioreactor. The experimental data showed that biomass and astaxanthin production of the second batch could reach the level of the first batch, no matter that the carbon source was glucose or hydrolysis sugar of starch. This result showed that this strain had good stability, and thus repeated batch and fed-batch process could be applied in astaxanthin fermentation for economical purpose.

Abstract:The knowledge of oxygen evolution characteristics, which is a symbol of photosynthetic activity, under various light conditions is important for photobioreactor design and operation. In this study, we constructed a device to investigate oxygen evolution characteristics of Spirulina platensis under two different light regimes: 1) continuous illumination of various light intensities (14?6 500 μmol/(m2·s)); 2) medium frequency L/D cycles of four different light intensities (69, 505, 1 330, 4 265 μmol/(m2·s)). Light limited region, intermediate region, light saturated region and light inhibited region of light intensity were recognized according to their relationship with oxygen evolution rate (OER) under continuous illumination. Investigation of S. platensis under L/D cycles showed whether photosynthetic efficiency could be increased with increasing L/D frequency largely depended on the light intensity applied. The higher the light intensity, the larger the photosynthetic enhancement could be expected with the increase of L/D frequency. The largest light integration effect was found under L/D cycles of high light intensity (4 265 μmol/(m2·s)) and medium light fraction (k=0.6), while light integration effect was totally absent under low light fractions (k<0.2). We also discussed their implications to the practical aspects of microalgae cultivation.

Abstract:We developed large-scale preparation of phycoerythrin from Porphyra haitanensis, a main economic red algae in China. Firstly, P. haitanensis thallus was broken by using “swelling and smash” method. Then times of grads ammonium sulfate precipitation applied to the crude extraction were compared. Desalted solution was further purified with one-step chromatography using hydroxyapatite and properties on spectrum and molecular weight were identified finally. The results indicated that after four times of ammonium sulfate precipitation (15%, 50%, 10% and 40%), the absorption spectrum purity of P. haitanensis achieved 0.9 (A564/A280), and 507.82 mg phycoerythrin (A564/A280>3.2) was obtained from 7 kg fresh algae after further hydroxyapatite chromatography. This research provides a potential way for preparation of phycoerythrin in large sclae.

Abstract:Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8?3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8?2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.

Abstract:In order to select better seeding sludge and promote start-up of Anammox reactors, we studied the start-up performances of three Anammox-EGSB bioreactors inoculated with anaerobic methanogenic sludge (AMS) (R1), Fresh Anammox sludge (FAS) (R2) and stored Anammox sludge (SAS) (R3), respectively. Results showed that these three seeding sludges could start up Anammox-EGSB bioreactors successfully, but the start-up progresses showed different characteristics. The start-up course of R1 could be divided into three phases including autolysis phase (15 d), lag phase (54 d) and activity elevation phase (40 d). However, the start-up courses of R2 and R3 only included lag phase (2 d and 12 d, respectively) and activity elevation phase (15 d and 57 d, respectively). Besides, the performance of R3 was better than that of R1, but worse than that of R2. Furthermore, bathing the Anammox sludge in the effluent of bioreactors was a convenient and effective way to keep the activity of the Anammox sludge. The ammonia removal efficiency, percentage of denitrification and the stoichiometric ratios of NH4+-Nr/NO2?-Nr and NO3?-Np/NH4+-Nr could serve as indicators to monitor the start-up of Anammox bioreactors.

Abstract:The aim of this study was to establish a fast and accurate method for developing specific DNA sequences and PCR primers for the detection of Staphylococcus aureus. An automatic C++ program for genomic comparison was used to identify specific DNA sequences from the genome of S. aureus MRSA 252. Four primer pairs were obtained from 9 specific target sequences by comparison of 2656 coding sequences with our local genome database, and 2 pairs of primers were confirmed to be specific to S. aureus by PCR evaluation against 137 bacterial strains, including 11 species of Staphylococcus. Furthermore, the DNA detection sensitivity of primer SA3 was 13.7 fg/μL and the cell sensitivity for this primer was 9.25×102 CFU/mL. This method has overcome the limitations of specific target mining in conventional assays, and it could be easily and widely used for other foodborne pathogens.

Abstract:In recent years, there are tremendous economic and social losses across the world because of virus-related diseases. It is well known that Madin-Darby canine kidney (MDCK) cells are easily handled, quickly amplified and efficiently infected with influenza virus. Therefore, they are considered as one of the most important cell lines for the production of influenza vaccine. In this work, we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM. Next, we derived a cell line named ssf-MDCK, which was amenable for single-cell suspension culture in the serum-free medium. We found that during serum-free batch culture of MDCK cells, the peak viable cell density and maximum specific growth rate were 3.81×106 cells/mL and 0.056 h?1, respectively; 3.6- and 1.6-fold increase compared with those in serum-containing adherent batch culture. In addition, we compared growth and metabolic characteristics of MDCK cells in serum-containing adherent culture, serum-free adherent culture and serum-free single-cell suspension culture. We found that less metabolic by-products were produced in both serum-free cultures. In serum-free single-cell suspension batch culture, the viable cell density was highest. These results are critical for establishing large-scale suspension culture of MDCK cells as subsequent well as large-scale influenza vaccine production.

Abstract:Elastin-like polypeptides (ELPs) are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin. It was a promising tag for recombinant protein purification. Here, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b(+). Then, we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl β-D-Thiogalactoside (IPTG), ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the [KV8F-20] decreased to 19 °C by 0.4 mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.

Abstract:We investigated the mechanism of human aspartyl β-hydroxylase (HAAH) in early diagnosis of tumors. The encoding gene of HAAH was cloned from the hepatic carcinoma by RT-PCR and expressed as a fused protein in the prokaryotic vector pBV-IL1. The expressed HAAH was purified by Ni2+-NTA purification column and the purified protein was then used to immunize Balb/c mice. Three hybridoma cell lines (respectively designated H3/E10, E4/F12 and G4/D8) stably expressing the monoclonal antibody specific to HAAH fusion protein were obtained. The specificity and sensitivity of the monoclonal antibody were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Finally, the monoclonal antibody expressed by H3/E10 cell line was used to detect the expression of HAAH in several tumor cell lines by indirect immuno-fluorescence, and the specific fluorescence was observed. In conclusion, this study successfully constructed the recombinant prokaryotic vector pBV-IL1-HAAH and prepared HAAH-specific monoclonal antibody for further study of the structure and function of the protein. The result may also lay solid foundation for the research of the molecular mechanism of HAAH in early diagnosis of tumors.