Abstract:The extremely acidophilic, obligately chemolithoautotrophic thiobacilli can obtain energy from the chemolithotrophic oxidation of inorganic-sulphur. They have industrial applications in metal leaching, desulfurization from coal and oil, agriculture, and environmental protection. However, their inability to use organic substance, their slow growth rate and low cell yield, has limited their further industrial use. The construction of engineered strains with better growth rate and improved ability to use organic compounds is important. In this paper, the inhibition of growth by organic compounds, limited use of organic compounds, central metabolic pathways, and transport mechanism of the extremely acidophilic obligately chemolithoautotrophic thiobacilli are reviewed, as well as the current research progress in their genetic modification to use organic compounds.

Abstract:Aluminum-containing adjuvants have been used over 80 years and play a very important role in many vaccines. However, the mechanism of aluminum-containing adjuvants as well as the problems existing in their application remain uncertain. HPAIV H5N1 arouses people’s attention and anxiety worldwide. Vaccination is an effective control measure. The use of aluminum hydroxide adjuvant will decrease the dosage of antigen whereas the vaccine reaches the same antibody titer. This paper summarizes some recent developments in HPATV H5N1 with aluminium-hydroxide adjuvant, and assesses the prospect of this new adjuvant vaccine.

Abstract:To improve the production of geldanamycin in Streptomyces hygroscopicus 17997, gene disruption was done to delete the naphthalenic AHBA genes (shnSOP), encoding the products that share the common biosynthetic substrates with geldanamycin. The resulting mutant strain (DSOP) was cultivated on a solid medium and the amount of spores collected from the plates was calculated from 5 to 14 days and the yield of geldanamycin was measured by HPLC. The geldanamycin production of the DSOP strain increased by 185% comparing with that of the parent strain. On solid medium, the DSOP strain underwent 2 cycles of sporulation and the growth of the second sporulation had the highest geldanamycin production.

Abstract:The antimicrobial peptide GK1, a derivative of Cecropin A, shows high antimicrobial activities against Gram-positive and Gram-negative bacteria. To avoid the lethal effects on Escherichia coli host cells during its expression, the GK1 gene was complexed with a modified human proinsulin (mhPI) gene, inserted into the vector pET28a to construct?the recombinant expression plasmid (pET28a-mhPI-GK1) and then transformed into BL21(DE3). A fusion protein was expressed and resulted in insoluble inclusion bodies counting 20% (W/W) of total cellular proteins. Recombinant GK1 was cleaved from the mhPI-GK1 fusion protein by cyanogen bromide (CNBr) and purified by cation exchange chromatography and reverse-phase HPLC. The final concentration of recombinant GK1 was 5.7 mg/L Escherichia coli culture and the purity was above 97%. Its molecular weight measured by Electrospray Ionizsation Mass Spectrometry (ESI-MS) was 2794 D, similar to that of the synthetic GK1. In addition, they have similar antibacterial activity. The results demonstrated that mhPI was capable of mediating high-level GK1 gene expression.

Abstract:Hypoxia inducible factor-1α (HIF-1a) is a transcription factor that responds to changes in oxygen concentration. In this study, we constructed two vectors, HIF-AB and HIF-CD, to transcribe functional short interfering RNA against different region of mouse HIF-1 a. The oligonucleotide encoding small hairpin RNAs against mouse HIF-1 a was inserted into the downstream of U6 promoter of pBSK/U6-NEO plasmid. Cre-expression vector CRE-ERT2 with either HIF-AB or HIF-CD was transfected into RAW264.7 cell line, after selection with G418 and hygromycin, to obtain cell lines with stabilized expression of CRE-ERT2 and HIF-AB, or CRE-ERT2 and HIF-CD. The expression levels of HIF-1α of these stable cell lines were detected by semi-quantitative RT-PCR following treatment of CoCl2, a HIF-1 a expression inducer. The HIF-1 a mRNA expression was reduced by 85% and 72% by HIF-AB and HIF-CD respectively. HIF-AB vector was microinjected into pronucleus of zygotes to generate transgenic mice. We got two founder and two first filial generation transgenic mice containing HIF-AB and these transgenic mice were crossed with EIIA-Cre transgenic mouse to get EIIA-Cre;HIFRNAiflox/+ mice. The conditional knock down mice were viable and developed normally. The results of RT-PCR indicated that the HIF-1α mRNA expression of liver, lung and kidney were reduced significantly compared with the normal control.

Abstract:Vitamin E, or tocopherol, is a lipid-soluble antioxidant and essential for human health. 2-methyl-6-phytyl-1, 4-benzo- quinol methyltransferase (MPBQ MT), one of the key enzymes in vitamin biosynthetic pathway in plants, converts 2-methyl- 6-phytyl-1,4-benzoquinol(MPBQ) to 2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ).In this study, we isolated the 1018bp promoter of Arabidopsis MPBQ MT gene. The promoter was fused with GUS reporter gene and this expression cassette was introduced into wild Arabidopsis by Agrobacterium-mediated transformation. GUS staining shows that GUS gene was expressed in stems, leaves, calyxes, stamens and siliques in transgenic Arabidopsis plants, with higher expression in green tissue such as stems, leaves, siliques, while invisible in roots, petals, and seeds. The data indicate that MPBQ MT gene promoter was likely to be expressed preferentially in green tissues such as stems, leaves, and young siliques.

Abstract:Ergosterol is economically important as the precursor of vitamin D2 and the main material to produce steroid drugs. The biosynthesis of sterols in yeast is complex. ERG6 gene encodes the sterol C-24 methyltransferase catalyzing the fifteenth reaction in ergosterol biosynthesis. In this study, ERG6 gene was cloned from Saccharomyces cerevisiae YSF-20 by PCR. To express ERG6 gene properly in S. cerevisiae, expression plasmid pPERG6 containing ERG6 under the control of PGK1 promoter was constructed and then introduced into S. cerevisiae YS58 to generate recombinant strain YS58(pPERG6). Results of ERG6 disruption and complementation showed that the ERG6 gene on plasmid pPERG6 expressed functionally in S. cerevisiae. The ergosterol content in recombinant strains YS58(pPERG6) was 1.32 times of that in the control strain YS58(YEp352), and at the same time, the biomass of the recombinant strain was 1.23 times of that of the control strain. Results in this study showed that the internal expression of ERG6 gene enhanced the ergosterol biosynthesis in yeast.

Abstract:We used Red recombination system to construct a modified E. coli protein-producing strain capable of autohydrolysing host nucleic acid. E. coli BL21(DE3), a common protein-producing strain, was used as starting material. The modified E. coli expres sion host had a staphylococcal nuclease expression cassette within the host chromosome lpxM locus. The Staphylococcus aureus nuclease was expressed as a fusion to the ompA signal peptide, and was translocated to the periplasm of the cell, protecting the host nucleic acid from the toxic activity during growth. The nuclease was released during cell lysis and subsequently hydrolyzed host nucleic acid in the lysate. Results show that the modified strain had sufficient nuclease activity to completely autohydrolyze the host chromosomal DNA and produced same amount of recombinant proteins as the original strain.

Abstract:Recombinant human interferon alpha (rHuIFNα) has been successfully used in antiviral and antitumor treatment. However, it degrades rapidly in vivo, resulting in the disappearance of cytokine in the plasma within several hours after administration. Strategies including chemical modification and fusion proteins have been used to improve the half-life of IFNα in vivo. In the present study, we constructed fusion proteins with IFNα2b and the IgG Fc region from various subtypes of human IgG and expressed them in methylotropic yeast, Pichia pastries. The fusion proteins were secreted to the culture medium as the active form of disulfide-linked homodimer with a single glycoslation modification on each molecule. Regardless of the subtype of Fcγ, fusing Fcγ fragments to IFNα2b showed impaired antiviral activities than the control IFNa2b in vitro. With the highest antiviral activity, IFNα2b-Fcγ2 was measured as 4.29×107 IU/mg, which decreased 2.3 times compared to the control IFNa2b. The half-life of IFNα2b-Fcγ2 in blood circulation extended to 65 hours and was still detectable until 120 hours, which was 8 times longer for circulation half-life and 10 times longer for metabolic kinetic time than the control IFNα2b. The results demonstrated that fusing IgG Fc region with IFNα2b could improve the pharmacokinetic properties of the fusion proteins, which has the potential in clinical therapy.

Abstract:We determined whether body-weight regulator, leptin, could influence differentiation of swine skeletal myoblasts. Myoblast occured in non-leptin and leptin formed in different concentrations (10~1000 ng/mL) for various duration (12~60 h). The morphologic assay demonstrated that leptin significantly decreased the formation of myotubes and myogenic index. In addition, biochemical analysis showed that leptin inhibited Creatine Kinase (CK) activity, expressions of myogenin and mitogen activated protein kinase (MAPK). As conclusion, exogenous leptin could inhibit differentiation via MAPK in swine skeletal myoblasts and this suggests that leptin be an important mediator in the regulation of muscle-cell differentiation.

Abstract:In this study, the basic sequences of 19peptide were synthesized and inserted into vector pTYB2. After being expressed and purified, the soluble 19peptide was obtained. The inhibiting effect on hepatocarcinoma cell was selected by 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay and cell growth curve. The apoptosis index was observed using TdT-mediated dUTP nick end labeling (TUNEL), flow cytometry and transmission electron microscope (TEM). Its anti-tumor activity in vivo was verified in H22 ascitic fluid transfevent hepatocarcinoma of mice. 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-di- phenyl-2H-tetrazolium bromide (MTT) assay and cell growth curve showed cell viability decrease with the 19peptide.Survival hepatocarcinoma cell decreased with time. In the treatment group, obvious change of apoptosis, the appearance of sub-G1 phase and dyed brown cells were seen by transmission electron microscope (TEM), flow cytometry and TdT-mediated dUTP nick end labeling (TUNEL), The inhibition rate of mouse H22 ascitic fluid transfevent hepatocarcinoma growth was 48.46%. As a whole, 19peptide could directly inhibit hepatocarcinoma cell proliferation and promote hepatocarcinoma cell apoptosis.

Abstract:Macroporous microcarriers (Cytopore) were used to construct microfabricated tissues. Perfusion approach was used to reassemble macrotissue on the basis of their aggregative property. Microfabricated tissues reached the maximum cell density with 16.4×107 cells/cm3 after 20 days. They were uniform with high cell viability and massive matrix as a whole after 32 days. The aggregation of the microfabricated tissues was visible especially in the later period of the culture, and a centimetre-size(12 mm × 6 mm)macrotissue with uniform cell and matrix distribution was obtained through perfusion reassembly. Constructing engineered tissues using macroporous microcarriers was effective and promising.

Abstract:We used biofunctional chromatography to study the interaction between insulin and immobilized insulin receptor(IR). The heterogeneous insulin receptor extract from Wistar mouse liver was immobilized onto the surface of an immobilized artificial membrane through coating. The detergent was removed by dialysis to reduce its interference with the chromatography. To optimize the protein immobilization, we optimized the coating conditions at: pH 7.2, ion strength 20 mmol, and organic solvent 2% (v/v). The competition and self-displacement interacting study of insulin with IR was conducted by the zonal elution using insulin as the self-competition agent, the result indicated that the insulin receptor was active and interacted with insulin. In addition, relative binding constant was determined by frontal analysis by changing the concentration of insulin in the mobile phase.

Abstract:We used high resolution two-dimensional polyacrylamide gel electrophoresis and ImageMaster 2D platinum techniques to study the total midgut proteins of silkworm, Bombyx mori in the 2nd, 5th and 7th day of the fifth instar stage. The characteristics of midgut proteomic differed from other reported silkworm tissues. Most protein spots were unevenly distributed in the area from 20 to 70 kD and PI between 4 and 8. Among them most spots located in acidic area, and this characteristic was observed in the map of midgut in the 7th day. There were 869 spots in the 2D map of midgut in the 2nd day, and 966 in 5th day. During this period, 97 new spots were generated, and mainly located in the area of pI 6-9 with molecular size between 20 and 40 kD. Along with larval maturation, the spots in the map decreased obviously, with 420 in the 7th day, 56.5% less than that of the 5th day. These results indicated that the proteins in the midgut had big change at the early, middle and late 5th stage, implying their adaptation in the larval midgut. Some spots were excised and identified by using MALDI-TOF MS, and they were involved in larval body structure, metabolism and immunity.

Abstract:Based on kinetic parameters analysis, a 4-stage glucose-control strategy was used to optimize pyruvate production by Torulopsis glabrata. At 0~18 h, 40 g/L glucose was fed to get a higher specific growth rate, and then glucose was quickly fed to the fermentation broth at 19~25 h to achieve the maximum dry cell concentration and to redirect the carbon flux from cell growth to pyruvate formation. At 26~63 h, a highest pyruvate yield (0.71 g/g) was achieved through maintaining glucose at 33.4 g/L by a singular control strategy. After 64 h, pyruvate fermentation was continued as batch process. A high concentration (83.1 g/L), yield (0.621 g/g) and productivity (1.00 g/(L·h)) of pyruvate were achieved by applying this strategy, which were 21.3%, 21.6% and 29.9% higher than that of the batch process.

Abstract:To study the effect of malic enzyme overexpression on succinate production in the pfl ldh double mutant Escherichia coli NZN111 (ldhA::Kan pfl::Cam) , we transformed the expression vector pTrc99a-sfcA into it and constructed the recombinant NZN111(pTrc99a-sfcA). The specific malic enzyme activity of the recombinant was 30.67 u/mg after 8-hour inducement by 0.5 mmol/L Isopropyl b-D-1-Thiogalactopyranoside, 140 times higher than that of NZN111. The two-step fermentation was used and the results showed that the overexpression of malic enzyme catalyzed the reverse reaction from pyruvate to malate, which was impossible under general conditions. Succinate accumulated as the major product. Cells at the late exponential phase were inoculated to an anaerobic fermentation with 0.7 mmol/L IPTG in medium containing 18.5 g/L glucose. The final concentration of succinate and acetate was 12.84 g/L and 0.58 g/L, respectively. Formate and lactate were not detected. The constructed metabolically engineered strain had the feature of higher succinate yield and less by-products.

Abstract:The nano-particles’ surface were modified by silane reagent Si(OC2H5)3C3H6NH2. Bovine serum albumin (BSA) was coupled by a series of glutaraldehyde concentration. Under optimal reaction conditions the maximum amount of immobilized BSA was 140 mg/mg Fe3O4, when glutaraldehyde concentration was above 10%. After immersing in anti-BSA rabbit serum and regenerating by Gly-HCl buffer, the purity of IgG by super-paramagnetic iron oxide (SIPO) nano-particles was characterized by UV-spectrometer, SDS-PAGE and immuno-diffuse. Results showed that subtotal separation was achieved at 10 min, 15mg SPIO in 1ml serum. The purity was much higher than that with the traditional (NH4)2SO4 method. The activity of special protein probe maintained above 78% after 5 consecutive uses.

Abstract:Bombyx mori baculovirus expression vector system (BEVS) was adopted to express S-hEGF. The reconstructed plasmids of pBacPAKS-hEGF and an engineering modified BmNPV Bm-BacPAK6 DNA were used to co-transfect BmN cells in order to obtain recombinant virus. The recombinant virus (named vBacPAKS-hEGF) was then used to infect BmN cultured cells, the fifth instars larvae and pupa. The aimed 12 kD protein expressed in BmN cultured cells and the fifth instars larvae was identified with Western blot. ELISA results showed that the amount of expression protein reached 23 mg/ 106 in BmN cells and 82 mg/mL in larvae. The biological activity was determined by epidermal growth factor dependent Balb/c3T3 cell line. Both cellular extracts and haemolymph of silkworm larvae infected with the recombinant virus accelerated proliferation of Balb/c3T3 cells. In addition, animal experiment results revealed that S-hEGF fusion protein promoted newborn ICR mice weight increasing, incisor eruption and eyelid opening.

Abstract:We report the production of recombinant human antithrombin Ⅲ (rhATⅢ) in transgenic cloned goats. Human ATⅢ mRNA was isolated from human liver tissues, then transcripted into cDNA. The human ATⅢ cDNA (without the first 96 base pair sequence)and Enterokinase recognised peptide-DNA sequence were ligated to a goat beta-casein promoter and poly(A) singling sequences, neomycin selection gene was linked at the end of the poly(A) singling region. The mammary expression construct was transfected into in vitro cultured goat foetal fibroblast cells and the cells were selected with 500ug/ml G418. Five transgenic cloned goats were derived by nuclear transplantation and their transgenic status were confirmed by Southern and/or PCR analysis. The first cloned goat died at 78 day old with abnormalities in respiratory system and kidney. Two of the male cloned goats were bred to produce female offspring. Milk analysis from the transgenic lines revealed that the size of the rhATⅢ in milk was about 60 kD. One line could produce rhATⅢ at 0.4 mg/L in their milk; another line could produce rhATⅢ at a large scale of 3 g/L in their milk. rhATⅢ was also a glycoprotein, which was similar to human serum ATⅢ.

Abstract:Expression of the Hc Fragment of Clostridium botulinum Neurotoxin Serotype A in Mammalian Cells with Recombinant Semliki Forest Virus Expression System

Abstract:To implement high cell density cultivation of transgenic Synechococcus sp. PCC 7002 with mouse metallothionein-I gene, and its applications in heavy metal wastewater, we optimized the medium components. We used response surface method, a useful tool for multi-factor process optimization, through a three- step experiment design: full factor design, the steepest climbing and central composite design. The selected three factors were NaHCO3, NaNO3, and initial pH. The optimized medium contained 1.696 g/L NaHCO3 and 8.57 g/L NaNO3, at initial pH 8.57. The optimized medium was validated in 2-L and 20-L air lift photo-bioreactor, respectively. The maximum cell concentration reached 4.16 g/L for 2-L bioreactor, and 3.12 g/L for 20-L bioreactor.

Abstract:We modified a previously constructed vector pOV1 and compared expression difference among modified constructs. First. 5￠-and 3￠-regulatory regions of chicken ovalbumin gene were excised from the previously constructed vector pOV1 by endonuclease digestion and subcloned into modified pcDNA3.0, named as pOV2. Then only the 5′-regulatory region was subcloned into the same vector and this resulted in the third oviduct-specific expression vector pOV3. To compare expression property of the three constructs in hen oviduct, the LacZ reporter gene was subcloned at the down-stream of the 5′-regulatory region in vectors pOV1, pOV2 and pOV3, respectively. The resultant recombinant constructs pOV1LacZ, pOV2LacZ and pOV3LacZ were injected into laying hens via wing vein rout. RT-PCR of the vector-injected hen tissues showed that the LacZ gene was transcribed only in the oviduct, but not in the heart, liver, kidney and spleen tested. Similarly, β-galactosidase activity was detected only in the oviduct magnum, which was secreted into egg white of the injected hens and enhanced by injecting estrogen into the hens. Among the three vectors tested, expression of LacZ gene in pOV3LacZ-injected hen oviduct magnum was at a relatively higher level and thus pOV3 vector was selected for further studies.

Abstract:We used Candiada sp. 99-125 lipase in bis (2-ethylhexyl) sulfosuccinate (AOT) reversed micellar systems to produce biodiesel from soybean oil. We studied the effects of solvent, concentration of AOT, ratio of water and AOT, buffer pH, temperature and rotating speed on the transesterification. Results showed that AOT reversed micellar systems provided a suitable micro-environment for biodiesel production. The optimal parameters were: the ratio of water and AOT 11, AOT concentration 50 mmol/L, temperature 40℃, pH 7, molar ratio of methano and oil 3, and rotating speed 180 r/min. The transesterification rate reached 90% when a 3-step methanolysis protocol was used.

Abstract:We examined the effects of three organic carbon sources on mixotrophic growth, biochemical components and fatty acid composition of Phaeodactylum tricornutum. Mixotrophically, P. tricornutum grew faster and had shorter doubling time. The biomass of P. tricornutum was greatly enhanced under mixotrophic condition with its highest biomass of 713 mg/L after 16 days, in medium containing 100 mmol/L glycerol. This was 1.60-fold of that obtained under autotrophic condition. The biomass during mixotrophic culture with 100 mmol/L glucose and 100 mmol/L acetate was 1.28-fold and 1.21-fold of that obtained under autotrophic condition, respectively. Compared with autotrophic condition, the content of soluble protein decreased obviously, whereas the content of soluble carbohydrate and total lipid increased. The content of lipid during mixotrophic culture with 100 mmol/L acetate and 100 mmol/L glycerol was 1.43-fold and 1.20-fold of that obtained under autotrophic condition, respectively. There was no difference between lipid content of mixotrophic growth with 100 mmol/L glucose and that of autotrophic condition. The eicosapentaenoic acid (EPA) content and yield was 6.23% and 36.59 mg/L during mixotrophic culture with 100 mmol/L acetate. These were 1.10-fold and 1.40-fold of that obtained under autotrophic condition, respectively. The EPA content and yield with glycerol and glucose were lower than that obtained under autotrophic condition. These results indicated that mixotrophic cultivation with acetate was beneficial to produce EPA.

Abstract:We optimized the chemical conjugation between R-phycoerythrin and antibody. First, the R-PE (R-phycoerythrin) was derived with beterobifunctional reagent SPDP(N-succinimidyl-3-2-pyridyldithio propionate) and antibody was thiolated with DTT(dithiothreitol). Second, we determined the effects of the different molar ratio of SPDP to R-PE for the derivation and DTT to IgG for the thiolation. The results showed that the optimum molar ratio of SPDP to R-PE for derivation was 40:1, and DTT to IgG for thiolation was 500:1. The conjugation between R-phycoerythin and antibody was further done for fluorescence probe preparation. R-phycoerythin was conjugated with IgG and formed a probe complex by whole wavelength scanning, electrophoresis (Native-PAGE) and fluorescence microscope observation.

Abstract:We used chromatography to remove endotoxin during the purification of rHSA-IFNα2b (recombinant human serum albumin-Interferon alpha 2b, rHSA- IFNα2b). Affinity chromatography of Blue-sepharose, hydrophobic interaction chromatography of SOURCE 15 ISO, ion exchange chromatography of Q Sepharose Fast Flow and gel filtration of sephadex G25 Coarse were applied consequently. The endotoxin levels were measured by Limulus Amebocyte Lysate gel-clot assay. Protein purity and concentration were determined by RP-HPLC. Up to 99.9% endotoxin of rHSA-IFNα2b was removed and to a concentration of 1 EU/mg. This process was effective to purify rHSA-IFNα2b and remove exdotoxin simultaneously.

Abstract:We observed the heterogeneity of consensus interferon (cIFN) expressed by Pichia Pastoris, such as aggregation and degradation. The effect of induction pH on degradation of cIFN was studied in a 5-L bioreactor. The heterogeneity of cIFN was less serious when induced at pH 4.0~5.0. The bioactivity of the fermentation supernatant reached 2.5×108 IU/mL when pH was controlled between 4.0 and 5.0. To seek the reason of cIFN degradation, we analyzed the activity of protease and cell mortality by flow cytometry. At lower pH, cell mortality was higher and more protease was released. Protease activity increased at higher pH. The proteolytic degradation of cIFN was enhanced at either high or low pH. The cIFN was degraded entirely when pH was 7.0, where the protease had its optimal pH.