Abstract:Animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host. The gut microbial community of humans and animals has received significant attention from researchers because of its association with health and disease. The application of metagenomics technology enables researchers to study not only the microbial composition but also the function of microbes in the gastrointestinal tract. In this paper, combined with our own findings, we summarized advances in studying gastrointestinal tract microorganism with metagenomics and the bioinformatics technology.

Abstract:Understanding inter-species transmission of influenza viruses is an important research topic. Scientists try to identify and evaluate the functional factors determining the host range of influenza viruses by generating the recombinant viruses through reverse genetics in laboratories, which reveals the viruses’ molecular mechanisms of infection and transmission in different species. Therefore, the reverse genetic method is a very important tool for further understanding the biology of influenza viruses and will provide the insight for the prevention and treatment of infections and transmission. However, these recombinant influenza viruses generated in laboratories will become the potential threat to the public health and the environment. In this paper, we discussed the biological safety issues of recombinant influenza viruses and suggested we should set up protocols for risk management on research activities related to recombinant highly pathogenic influenza viruses.

Abstract:The study aims at cloning the CDS fragment of erk2 gene cDNA in Inner Mongolia Cashmere Goat and analyzing its tissue-specific expression. erk2 gene cDNA was cloned by RT-PCR. The nucleotide sequence was analyzed by Blast and amino acid sequence was analyzed by online softwares SMART and Psite. The tissue-specific expression pattern of erk2 was analyzed by quantitative RT-PCR. The expression of erk2 in testis of goat was detected by Immunohistochemistry. The cloned erk2 gene cDNA (GenBank Accession No. JX569765) was 1 083 bp in length, including a complete ORF encoding 360 amino acids residues. The amino acid sequence shares 100% identity with the Bos Taurus ERK2 (Bos Taurus BC133588.1). Analysis by SMART suggests that the encoded protein contained a “TEY” structure and an S-TKc domain possessing serine/threonine kinase catalytic activity. Analysis with Psite indicates one cAMP-/cGMP-dependent protein kinase phosphorylation site, 3 protein kinase C phosphorylation sites, 5 casein kinase II phosphorylation sites, 2 protein kinases ATP-binding region signatures and one serine/threonine protein kinases active-site signature in this protein. Analysis by Psort (k-NN prediction) suggestes that this protein most probably is localized in cytoplasm. The results of quantitative RT-PCR show that the expression of erk2 mRNA was higher in heart, skin and breast, whereas lower in spleen and kidney. ERK2 protein was detected in testis by immunohistochemistry.

Abstract:Directed evolution was conducted to improve the thermostability of lipase from Rhizopus chinensis CCTCC M201021. Mutations were introduced by two rounds of error-prone PCR and mutant lipase was selected by fast-blue RR top agar screening. Two positive variants were selected in the first-round and four in the second-round screening process. Ep2-4 was proved as the most thermostable lipase and its DNA sequencing revealed three amino acid substitutions: A129S, P168L and V329A. Compared with the parent, its half-life at 60 °C was 5.4- times longer and T50 was 7.8 degrees higher. Purified lipase of Ep2-4 was characterized and the result shows that its thermostability improved without compromising enzyme activity. According to the mimicked protein structure, mutation A129S formed a hydrogen bond with Gln133 and improved the thermostability by increasing the hydrophilicity and polarity of protein; mutation P168L by forming a hydrophobic bond with the nearby Leu164.

Abstract:Although DNA vaccination is now a promising strategy against hepatitis B virus (HBV) infection, this approach has relatively modest antiviral effect, indicating that immunosuppressive mechanisms may occur in the long-term established infection. In this study, we studied the immunogenicity and anti-HBV efficiency of a combination of HBV surface (HBsAg) and core (HBcAg) DNA vaccine, enhanced by heat shock protein (HSP) gp96 or HSP70 and mediated by in vivo electroporation. Immunization with gp96 adjuvanted HBsAg/HBcAg DNA formulation induced potent T cell and antibody immunity against HBsAg and HBcAg. Notably, treatment with gp96 or HSP70 as adjuvant resulted in reduction of Treg populations by around 20%. Moreover, compared with nonimmunized control mice, immunization with gp96 or HSP70 adjuvanted DNA vaccine dramatically decreased serum HBsAg and viral DNA levels, and HBcAg expression in liver. These results may therefore provide an effective strategy for designing gp96-based DNA vaccine for immunotherapy of chronic HBV infection.

Abstract:To study the expression of herpes simplex virus type 2 latency-associated transcript (LAT) open reading frame 1 (ORF1) and its anti-apoptosis function induced by actinomycin D in Vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, and the expression of ORF1 was identified by RT-PCR. The changes of Vero cells morphology induced by actinomycin D were observed by fluorescence microscopy, Hochest33258 fluorescence staining. Cells viability was evaluated by MTT assay and cells apoptosis rate was detected by flow cytometry. Double digestion and sequencing confirmed the pEGFP-ORF1 was constructed successfully, RT-PCR showed that the target gene was highly expressed in Vero cells. Hochest33258 staining reaveals that Vero cells transfected with pEGFP-ORF1 and induced apoptosis by actinomycin D had no changes in morphology. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmid pEGFP-ORF1 and induced apoptosis by actinomycin D has no statistically significant difference compared with the untreated normal control group (P>0.05), but remarkable higher than Vero cells transfected with empty plasmid pEGFP-C2 and induced apoptosis by actinomycin D, the difference was statistically significant (P<0.05). Flow cytometry assay shows that the cells apoptosis rate had no significant difference between pEGFP-ORF1 group and the normal group, but the cells apoptosis rate of pEGFP-ORF1 was lower than the pEGFP-C2 group. HSV-2 LAT ORF1 gene can be expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.

Abstract:Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this MIGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.

Abstract:The basement membrane (BM) is crucial in regulating the physical and biological activities of esophageal epithelial cells which attach to the underlying BM. In order to simulate the natural construction of BM, we prepared the fibrous scaffolds using biodegradable polylactide (PLA) and silk fibroin (SF) as the materials via electrospinning technology. BM’s proteins containing collagen (IV), laminin, entactin and proteoglycan were extracted from porcine esophagus and coated on the eletrospun fibers. Morphology, mechanical strength, biodegradability and cytocompatibility of the coated and uncoated scaffolds were tested and evaluated using scanning electron micrography, mechanical test system, immunofluorescence assay and western blotting with CK14 as the primary antibody. The fibrous scaffold PLA or PLA/SF, generated from the present protocol had good formation and mechanical and biodegradable properties. After coating with BM’s proteins, the scaffold could enhance the growth and differentiation of esophageal epithelial cells, which would contribute to remodel and regenerate the tissue engineered epithelium and further contribute to engineer the whole esophagus in future.

Abstract:Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17–20 mg/L with viable cell density at 7×106–10×106 cells/mL.

Abstract:To study the angiogenic activity of amphoteric brush-type copolymer complex of alginate-graft-PEI/pVEGF (Alg-g-PEI/pVEGF) in vivo, we evaluated the toxicity of Alg-g-PEI/pVEGF complexes to rMSCs and zebra fish first. Then, we used gel retardation assay to investigate the protection of complex to pDNA against DNase I, serum and heparin. For in vivo study, we evaluated the angiogenic activity of Alg-g-PEI/pVEGF complexes by using CAM and zebra fish as animal models, PEI 25K/pVEGF and saline as positive and negative controls. Our results show that Alg-g-PEI protected pVEGF from enzymolysis and displacement of heparin in some degree, and its complexes with pVEGF were less toxic to rMSCs and zebra fish. Alg-g-PEI/pVEGF complexes induced significant angiogenesis, which was dosage-dependent. In CAM, when the dosage of pVEGF was 2.4 μg/CAM, Alg-g-PEI group achieved the maximum of angiogenesis, and the area ratio of vessel to the total surface was 44.04%, which is higher than PEI 25K group (35.90%) and saline group (24.03%) (**P<0.01). In zebra fish, the angiogenesis increased with the increase of N/P ratios of Alg-g-PEI/pVEGF complexes in our studied range; when N/P ratio was 110, the optimal angiogenesis was obtained with vessel length of 1.11 mm and area of 1.70×103 pixels, which is higher than saline group (0.69 mm and 0.94×103 pixels) (**P<0.01) and PEI 25k group (0.82 mm and 1.11×103 pixels) (**P<0.01). Our results demonstrate that Alg-g-PEI/pVEGF significantly induces angiogenesis in CAM and zebra fish, and has a great potential in therapeutic angiogenesis.

Abstract:To capture a state of the enzyme in complex with an intact substrate, we developed and adopted a novel freezing method in crystal preparation procedure. Neither the elimination of the catalytically indispensable ligands, nor mutation or modification of the active site is required. At -20 ℃, we soaked the crystal of 6-phosphate-β-glucosidase (BglA) in the liquor containing p-nitrophenyl-β-D-glucopyranoside-6-phosphate (pNPβG6P). The diffraction data at 2 ? resolution was collected and an intact and unambiguous electron density map of pNPβG6P was obtained. These results provide an effective method for the research of cryoenzymology and the intermediate state of enzyme-substrate complex in the future.

Abstract:In order to study the effects of Ca2+ in the biosynthesis of salvianolic acid B (Sal B) induced by salicylic acid (SA) in the young seedlings of Salvia miltiorrhiza, we used confocal laser scanning microscopy and high performance liquid chromatography to measure the change of relative fluorescence intensity of Ca2+ and the contents of Sal B induced by SA before and after the application of extracellular calcium channel inhibitors (VP and LaCl3), intracellular calcium channel inhibitor (LiCl), as well as intracellular calmodulin antagonist (TFP). SA could induce the calcium burst, and the Ca2+ peak could last to 2–3 min in the guard cells of S. miltiorrhiza, which prompted the biosynthesis of Sal B after the Ca2+ burst. Both Vp or LaCl3, and LiCl or TFP could inhibit the burst of Ca2+ and the biosynthesis of Sal B. The above results demonstrated that Ca2+ from the extracellular and the intracellular calcium store regulate the biosynthesis of Sal B elicited by salicylic acid in S. miltiorrhiz young seedlings.

Abstract:In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.

Abstract:Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Abstract:Bacillus amyloliquefaciens B10-127 was used to produce 2,3-butanediol (2,3-BD) from residual glycerol obtained from biodiesel synthesis. Important variables for 2,3-BD fermentation, pH and dissolved oxygen, were studied. When pH was maintained constant, the yield of 2,3-BD was inhibited. The highest 2,3-BD yields were achieved by fermentation without any pH control with an optimized initial pH 6.5. Batch fermentative production of 2,3-BD by B. amyloliquefaciens was investigated using various oxygen supply methods by changing agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific 2,3-BD formation rate (qp), a three-stage agitation speed control strategy was proposed, aimed at achieving high concentration, high yield and high productivity of 2,3-BD. Maximum concentration of 2,3-BD reached 38.1 g/L, with the productivity of 1.06 g/(L·h), which were 14.8% and 63.1% over the best results from constant agitation speeds. In a pulse fed-batch fermentation, 2,3-BD concentration and productivity were significantly improved to 71.2 g/L and 0.99 g/(L·h), respectively. To our knowledge, these results were the highest for 2,3-BD production from biodiesel-derived glycerol.

Abstract:Nitrogen source is one of the important factors that affect the microalgae growth and lipid accumulation. We studied the effects of various nitrogen sources (i.e. NaNO3, CO(NH2)2, NH4Cl and CH3COONH4) and amount on the growth density, lipid yield, and eicosapentaenoic acid (EPA) content of Nannochloropsis oculata by single factor experimental method. The results show that N. oculata preferred NH4+ as nitrogen source rather than NO3- and CO(NH2)2. NH4+ could promote the growth and lipid accumulation of N. oculata. With the increase of nitrogen concentration, the biomass and the content of polyunsaturated fatty acid (PUFA) increased, but the content of lipid decreased. CH3COONH4 was the most suitable for growth, accumulation of lipid and EPA of N. oculata among the four investigated nitrogen sources. The optimal concentration was 5.29 mmol/L.

Abstract:Biosurfactants (BSs) are highlighted owing to their multiple advantages in diverse applications. To screen a superior strain that producing a blend-biosurfactant of lipopeptide and glycolipid, the hemolytic activity assay on blood agar plates, the modified oil-red spreading test and MALDI-TOF Mass Spectrometry identification of the purified products was carried out. Bacillus subtilis THY-7 was selected and its principal products were surfactin and dirhamnolipid. The medium component and culture conditions of THY-7 were optimized by both single factor and orthogonal experiments. After 48 h optimal batch culture in flask, the cell density (OD600) was 37.0 and the product titer was 2.4 g/L, which was 3.4 folds and 3.1 folds of that under original condition, respectively. A fed-batch culture in a 5 L fermentor was further performed coupling with in situ recovery of foam, in which the titer of blend-BS increased to 4.5 g/L at 25 h. Quantification by HPLC and anthrone colorimetry revealed that surfactin and dirhamnolipid accounted for 74% and 22% of the blend-BS, respectively.

Abstract:During the anaerobic fermentation by Escherichia coli AFP111 for succinic acid production, the viable cell concentration and productivity were decreased with the raising of succinic acid concentration. In order to restore cellular succinic acid productivity and prolong fermentation time, we collected strains and refreshed medium for repetitive succinic acid production. However, productivity is lower than that in the anaerobic fermentation before reusing strains. To enhance the productivity, strains were aerobically cultivated for 3 h in pure water before anaerobic fermentation. The activities of key enzymes were enhanced for better performance in producing succinic acid at anaerobic stage. After three rounds of repetitive fermentations, succinic acid concentration and yield reached to 56.50 g/L and 90% respectively. The succinic acid productivity was 0.81 g/(L·h), which was 13% higher than the repetitive fermentations without aerobic activation of the strains.

Abstract:With the advantage of clear target and little side effect, antibody drug has attracted widely attention of worldwide pharmaceutical companies. However, large scale mammalian cell culture and antibody quality analysis are the bottlenecks of antibody drug industrialization in China. Especially due to the significant effect of cell culture conditions on antibody heterogeneity. Therefore, it is extremely urgent to optimize cell culture conditions to favor the demands of antibody drug development. This review summarized the most recent advances in the effect of cell culture conditions on antibody quality, followed by addressing the key issues that might be strategically important for domestic antibody drug development.