Abstract:Embryonic stem cell is promising for regenerative medicine. However, its application is hampered by the utilization of eggs in most established methods. Recently, a new pluripotent stem cell establishing method was reported that, mouse and human differentiated cells could be induced reprogrammed into a pluripotent state by expressing exogenetic stem factors such as Oct4, Sox2, et al, through retroviral transduction. This approach avoiding egg use is a great breakthrough not only in stem cell technology but also present theory hypothesis of reprogramming. Here these works were reviewed in this article. Both the mechanism of induced reprogramming and the prospects of induced pluripotent stem cells were discussed.

Abstract:We identified the critical amino-acid residues in antigen M derterminant(MAD) epitope of human cytomegalovirus protein M. On the basis of the peptide sequence of MAD, some conservative residues were mutated into the glycine residue. Then the gene fragment of mutants linked to amino terminal of Fc were cloned into the plasmid pET32-Fc and expressed by fusion with Fc. After purified by protein A affinity chromatography, the activity of mutants binding the goat polyclonal antibodies against human cytomegalovirus(HCMV) were detected by ELISA and Western blotting. Our results showed that when glutamine residue was mutated into glycine residue, the activity of MADQ→G binding the goat polyclonal antibodies against HCMV was reduced apparently. Other mutants did not have the same characteristics. The activity of MAD was closely related to the conformation of glutamine residue.

Abstract:Geldanamycin (Gdm), an inhibitor of heat shock protein 90 (Hsp90), shows antitumor and antivirus bioactivity. Most Geldanamycin biosynthetic genes have been cloned from the genome library of Streptomyces hygroscopicus 17997. In this report, polyketide synthase (pks) gene, mono-oxygenase (gdmM) gene and carbamoyltransferase gene (gdmN) were subjected to inactivation. Three gene disrupted mutants (△pks, △gdmM and △gdmN) were obtained by double crossover. No Geldanamycin production was detected in three mutant strains cultured in fermentation broth. Gene complementation experiments excluded the possible polar effect of gene disruption on other genes. These results confirmed that pks, gdmM and gdmN genes were essential for Geldanamycin biosynthesis.

Abstract:The comparative sequence analysis is the most reliable method for RNA secondary structure prediction, and many algorithms based on it have been developed in last several decades. This paper considers RNA structure prediction as a 2-classes classification problem: given a sequence alignment, to decide whether or not two columns of alignment form a base pair. We employed Support Vector Machine(SVM) to predict potential paired sites, and selected co-variation information, thermodynamic information and the fraction of complementary bases as feature vectors. Considering the effect of sequence similarity upon co-variation score, we introduced a similarity weight factor, which could adjust the contribution of co-variation and thermodynamic information toward prediction according to sequence similarity. The test on 49 Rfam-seed alignments showed the effectiveness of our method, and the accuracy was better than many similar algorithms. Furthermore, this method could predict simple pseudoknot.

Abstract:We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe’s concentration, Mg2+ concentration, primers concentration and sample DNA extraction, rea1-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10×101 copies/mL and 10×108 copies/mL, and the detection limit of FQ-PCR is 1.0×101 copies/mL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.

Abstract:Human arrest defective 1(hARD1) is an acetyltransferase; its physiological significance remains unclear. To explore the relationship between ARD1 protein and tumors, we detected the hARD1 protein in tumor tissues in vivo. We cloned hARD1 gene from Hela cell and construct recombinant plasmid pET28b-hARD1. The recombinant plasmid was transformed into E. coli BL21(DE3)plysS. hARD1 protein was expressed by inducing with IPTG(1 mmol/L) and purified up to 95% through Ni2+ chelation affinity chromatography. We used the purified hARD1 protein as antigen immunized the Balb/c mice and obtained the hARD1 specific polyclonal antiserum. Through immunohistochemical analysis of different tumor tissues in vivo, we found that hARD1 expressed at high frequency in breast cancer, prostate cancer and lung cancer, especially, hARD1 expression frequency in breast cancer was up to 70%, which is higher than in the other tumors. These results indicate that the high expression level of hARD1 could be an indicator of the breast cancer. This new finding would be a foundation to further explore the relationship between breast tumor and hARD1.

Abstract:Cell adhesive molecular P-selectin was cloned, expressed and anchored on CHO cell membrane through GPI for selection specific antibodies. Total human platelet RNA was extracted and the functional segment of P-selectin gene was cloned by RT-PCR. The P-selectin functional segment gene was cloned into a eukaryotic expression vector pMCEw2-GPI containing an attenuated neo gene together with a downstream GPI, which was synthesized by overlapping PCR. The recombinant plasmid pMCEw2-GPI-P- selectin was then transfected to CHOdhfr- cells and screened with G418. ELISA, western-blot and immunofluorescence were carried out to detect the stability of P-selection expression on cell membrane. These results provided a necessary basis for the following study of selection the antibodies targeting P-selectin.

Abstract:This study aimed to obtain recombinant fusion protein of thymosin alpha1(TM-a1) and consensus IFNa (IFNa-con) which have bath TM-a1 and IFNα-con activities. The DNA sequence for the fusion protein was cloned into expression vector of pET-22b (+) and expressed in BL21 (DE3)-Codon plus-RP-X. The expressed product (TM-a1-IFN-con) was soluble, and amounted to more than 20% in total proteins of E. coli. By precipitation of (NH4)2SO4, hydrophobic interaction chromatography (HIC, Phenyl Sepharose 6 Fast Flow), anion-exchange chromatography (Q Sepharose Fast Flow), cation-exchange chromatography (SP Sepharose Fast Flow) and gel filtration (Sephadex G-75), it was purified to more than 96% purity. The activity of fusion protein for antivirus was tested by cytopathic-effect inhibition assay and activity for promoting lymphocyte proliferation is tested by cell proliferative assay. The activity for antivirus was higher than commercial IFNα1b and IFNα2a and activity for promoting lymphocyte proliferation was similar to commercial TM-α1. The fusion protein had better effect for anti-HBV in vitro, its effect was stronger than combination of IFNa and TM-a1 and cell toxicity was less than combination of IFNa and TM-a1. The above results show that it has effect bath antivirus of IFNa and promoting lymphocyte proliferation of the soluble fusion protein expressed in E. coli.

Abstract:Pretreatment with brain-derived neurotrophic factor(BDNF) reduces ischemic damage after focal cerebral ischemia, and bone marrow mesenchymal stem cells(MSCs) were reported to ameliorate functional deficits after stroke in rats. Here we investigate the synergistically therapeutic effects of BDNF gene-modified MSCs on cerebral infarction. We transfected MSCs with the BDNF gene using a lentivirus-based system and investigated whether the BDNF-modified MSCs contributed to improved functional recovery in a rat transient middle cerebral artery occlusion(MCAO) model. Compared to untreated rats, rats that received both MSCs and BDNF-MSCs showed significantly more functional recovery. The difference in modified neurological severity score(mNSS) was statistically significant(P<0.001). Recovery was better in BDNF-MSCs than in MSCs(P<0.001). At the second week and second month after the systemic delivery of blank vector-modified MSCs and BDNF-modified MSCs, the treated rats exhibited more significant recovery than the control, including the accumulation and living of enhanced green fluorescence protein(EGFP)-positive cells in the infarct area and surrounding areas, neuron-like changes, expression of surface markers of neural cells, and a large amount of BDNF expression in the BDNF-MSCs-treated group. Our findings suggest that BDNF-gene-modified rMSCs can migrate to surrounding areas of the cerebral infarction lesion, differentiate into neural cells, and survive for extended periods. With the synergy of BDNF, they may promote the recovery of the neurological function following cerebral infarction and represent a new strategy for stem cell-based therapy.

Abstract:Mechano-growth factor (MGF) is one of IGF-1 isoforms. MGF is mechanosensitive and has important functions in muscle hypertrophy, regeneration and nerve injury recovery. In this study, MGF cDNA (330 bp) was cloned from stretched osteoblasts by RT-PCR. In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5′ end sequence was truncated by primer, then the obtained truncated MGF (des(1-3)MGF) cDNA (321 bp) was subcloned in pET32a(+) vector to construct a prokaryotic recombination expression plasmid. Trx/des(1-3)MGF fusion protein, existing in forms of solution, was expressed in transformed Escherichia coli strain BL21(DE3) by IPTG induction at 30oC. The supernatant of cell lysates was subjected to ion exchange chromatography and Ni2+ metal affinity chromatography, and the fusion protein was obtained with the purity over 95%. After the fusion protein was cleaved by enterokinase, Trx and des(1-3)MGF was isolated by reverse-phase HPLC. Through these procedures, des(1-3) MGF was obtained with the purity of 98%. The protein molecular mass was conformity to the theoretical value by SDS-PAGE and mass spectrometry analysis. The purified des(1-3)MGF was incubated with MC3T3-E1 for cell proliferation and migration assays. The results show that des(1-3)MGF exhibited more facilitative effects on proliferation and migration of MC3T3-E1 than that of des(1-3)IGF-1.

Abstract:Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZaA, then the yeast expression vector pPICZa-hK1 was constructed. After transformed into Pichia pastoris host X33, high-level expression transformants were screened by escalating the concentration of Zeocin (from 500 to 700 mg/mL) of YPD plate and medium. When temperature was 30oC, pH 6.0 with induction duration of 64 hours in the 30 L fermenter, the highest yield can reach about 6500 u/L (1.25 g/L). The variation of glycosylation resulted in two kinds of molecules, i.e. rhK1-H with a heavy molecular weight and rhK1-L with a light one. rhK1 was purified from the supernatant through Phenyl hydrophobic interaction, Cu2+-charged Chelating and Anion-exchange chromatography. 0.28 g rhK1-H and 0.62 g rhK1-L can be purified from one liter supernatant. The yield recovery was 72% with a purity of >96%. So far our yield of rhK1 is superior than known recombinant expression method reported by other researchers.

Abstract:According to the amino acids sequence of OC-IΔD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IΔD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IΔD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IΔD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IΔD86. OC-IΔD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl β-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IΔD86:6His gene accounted for 11.4% of total protein and 16.4%of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.

Abstract:Enzymatic preparations obtained from young plants and cell cultures of capulin were screened for hydroxynitrile lyase activity. The threeweek old plants, grown under sterile conditions, were used to establish a solid cell culture. Crude preparations obtained from this plant material were evaluated for the transformation of benzaldehyde to the corresponding cyanohydrin (mandelonitrile). The results show that the crude material from roots, stalks, and leaves of young plants and calli of roots, stalks, internodes and petioles biocatalyzed the addition of hydrogen cyanide (HCN) to benzaldehyde with a modest to excellent enantioselectivity.

Abstract:In order to obtain salt-tolerant calli of Dandelion (Taraxacum officinale Weber), calli were induced from leaf explants of Dandelion on Murashige and Skoog’s medium supplemented with 2.0 mg/L 6-benzyladenine and 0.5 mg/L 2,4-dichlorophen oxyacetic acid With 1.5% NaCl as selection pressure, most calli became brown and dead, whereas some new cell clusters appeared at the edge of the brown calli after 2 to 3 weeks. The survived cells were picked out and sub-cultured every 3 weeks onto the fresh selection medium and salt-tolerant calli were finally obtained through 4 continuous selections on the selection medium supplemented with 1.5% NaCl. Salt-tolerant calli increased steadily under a fixed NaCl stress though their relative growth rate decreased with increased NaCl concentration whereas the control calli which were sub-cultured by 4 continus selctions on salt free medium ceased to grow under the same condition. This result indicated that the salt-tolerance of the selected calli is improved and this character is stable. Compared with the control, the SDS-PAGE pattern of the salt-tolerant calli had a unique 34 kD protein band. Its 30 kD and 18 kD protein bands were up-regulated. Further more, within the NaCl stress range up to 1.5%, the activities of antioxidant enzymes such as super oxidase dimutase, peroxidase and catalase, and the proline contents of the salt-tolerant calli were higher than those of the control. The results indicated that the selected calli with improved and stable salt tolerance were cell variants. The accumulation of the organic compatible solutes including proteins and the enhanced antioxidant capabilities in the salt tolerant calli are the two ways for them to regulate their osmotic homeostasis and alleviate the secondary reactive oxygen spexies damage respectively.

Abstract:Obtaining human blastocysts is a prerequisite for cell replacement therapy using embryonic stem cells. We established an interspecies somatic cell nuclear transfer (iSCNT) technique for producing blastocysts without sacrificing human oocytes. Human foetal fibroblasts were used as donor cells injected into the enucleated bovine oocytes in nuclear transfer, whereas bovine foetal fibroblasts were used to produce intraspecies embryos. We also examined the fate of human and bovine mitochondrial DNA (mtDNA) during preimplantation development after nuclear transfer by PCR. PCR analysis for the detection of human and bovine mtDNA was done at the 2,8-morula, and blastocyst stages of the embryos. Result: 2.8% interspecies embryos developed to blastocysts after cultured in an SOF medium, while blastocyst rate of intraspecies embryos were 10.1%. Both human and bovine mtDNAs existed until the morula stage, whereas only the bovine mtDNA was found at the blastocyst stage. These results indicated that interspecies cloning without using human oocytes could generate human blastocysts. Because of the incoordination between bovine mtDNA and human nuclear gene, developmental rate of interspecies embryos was significantly lower than intraspecie. Whether the embryonic stem cell could be used for cell replacement therapy need further research.

Abstract:To determine the direct effect of leptin on adipose tissue apoptosis in vitro using rat adipose-derived stem cells(ADSCs), we isolated the ADSCs of rat epididymis adipose tissue by collagenase digestion, filtration, and subsequent centrifugation. Cell cultures with or without leptin (10-9 mol/L, 10-8 mol/L, 10-7 mol/L and 10-6 mol/L) were incubated for different time. We examined the cell surface phenotype by immunofluorescence and detected the apoptosis morphological changes of ADSCs by laser scanning confocal microscope(LCSM). The number of apoptotic cells was determined by flow cytometry assay after annexin V binding and PI staining. Caspase-3 activity was measured by spectrofluorometry. The present study demonstrates that leptin treatment causes a marked increase in adipose-derived stem cell apoptosis. With the LCSM, after being treated with leptin, ADSCs showed the typical characteristic of apoptosis. Leptin in used concentrations (0 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L) caused a marked increase in cell apoptosis after 48 h incubation time (for 2.50%±0.72%, 6.78%±1.99%, 11.99%±1.58% and 17.93%±4.82%, respectively, P<0.05). Caspase-3 activity increased and reached a maximal level after 48 h in a linear fashion. The effect of leptin was dose-dependent and time-dependent. Leptin has been demonstrated to induce preadipocyte and adipocyte apoptosis, and today we demonstrate that leptin can induce ADSCs apoptosis, which can contribute to the decrease of adiposity. To our knowledge, this is the first study demonstrating the direct peripheral effect of leptin on ADSCs.

Abstract:Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosaccharides with long chains from xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increase in the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19. The addition of D-glucose to the culture of the mutant strain U-3 of B. pumilus X-6-9 repressed the synthesis of b-xylosidase, but not xylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharides with long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized. The hydrolyzates generated by the purified xylanase contained xylobiose, xylotriose, xylotetraose, and xylopentaose, but not xylose.

Abstract:Jingzhaotoxin-V(JZTX-V) isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion(DRG) neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS) and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.

Abstract:A novel anaerobic fermentation process—multistage countercurrent fermentation was applied to improve the bioproduction of volatile fatty acids (VFAs) from excess municipal sludge. Results showed that the total VFAs concentration and the total VFAs yield reached (10.5±0.5) g/L and 0.20 gVFAs/gVS(Volatile solid) using this novel process. Comparing with the conventional anaerobic fermentation, the concentration and yield of total VFAs increased by 31% and by 54%, respectively. Moreover, removal ratio of organic solids also increased by 37% and it was 50% at the end of multistage countercurrent fermentation. We further investigated the mechanism of VFAs production. Results revealed that this novel process could reduce the inhibitory effect of VFAs on the acid-forming microorganisms, and the total VFAs yield and the removal ratio of organic solids respectively depended on the first stage and the third stage of this novel process. Therefore, the multistage countercurrent fermentation can efficiently improve the bioproduction of VFAs from excess municipal sludge, and relatively enhance the removal ratio of organic solids.

Abstract:The anaerobic ammonium oxidation was successfully started up within 144 d in attached microbial film expanded bed reactor with bamboo carbon as carriers. The maximum nitrogen removal rate reached 3.02 kgN/m3/d, which was the highest level in China. The reactor has a great potential in nitrogen removal compared to the theoretical value 12.77 kgN/m3/d deduced from dynamic fit. As the start-up course experienced autolysis phase, activity lag phase and activity elevation phase, the seed sludge turned from khaki flocs to sandy brown granules and finally to red granules. These red granules with a relatively high anaerobic ammonium oxidation(ANAMMOX) activity of 0.56 mgTN/(mg protein)/h were major contributors to the ANAMMOX process in the reactor.

Abstract:We found seven tag sequence with high homology in dbEST by using human musclin gene, and got its cDNA sequence, which consists of 651bp and the open reading frame was 54~452 bp detected by RT-PCR, encoding 132 amino acid residue protein . The new gene has high homology with that of human, mouse and rat, the rate is 87.2%, 77.6% and 77.9%, respectively; the gene fragment was cloned into expression vector pGEX-4T-1, and the recombinant was transformed into E. coli BL21. Induced by IPTG, the fusion protein GST-musclin, a 38.59 kD protein was successfully expressed in E. coli BL21 and identified by Western blotting.

Abstract:In this article, a cell culture microchip was fabricated on the SU-8 mold based on polymer-MEMS process. In the microchip, the cell culture area was separated with microchannel by a microgap, which kept the cell culture area independent, but also regulated the micro-environment of extracellular matrix by the microfluidic flow. The cell culture microchip provided a new platform for cell research.

Abstract:Equine interferon-gamma (eIFN-g) expressed both in E. coli and baculovirus were evaluated for antiviral activity against recombinant Vesicular Stomatits Virus expressing green fluorescence protein (rVSV-GFP) in EFK-78 cells. The assays were conducted in 96-well plate. Virus infectivity was measured by quantifying GFP-positive cells, instead of quantifying the CPE reduction. Prior to infection of EFK-78 cells with rVSV-GFP, the cells were incubated with eIFN-g. The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with eIFN-g in a dose-dependent manner, comparing with the mock-treated cells. The titers of antiviral activity were 1×103AU/mL and 1×105AU/mL of eIFN-g expressed from E. coli and baculovirus, respectively. The antiviral activities of the recombinant eIFN-g were highly efficient and specific, as it was blocked by mAbs against eIFN-g.

Abstract:The avirulent QU strain of fowl adenovirus, a member of duck adenovirus type 1, could be a potential vector in recombinant vaccine development. To identify a non-essential region for replication of QU virus, a 3.4 kb fragment near the E4 region of QU virus genome was amplified by PCR to construct a plasmid pADGFP, in which ORF1, ORF8 and ORF9 was replaced with a system expressing enhanced green fluorescence protein. Further, a recombinant virus rQUGFP was constructed by homologous recombination after pADGFP and QU virus were co-transfected into chick embryo fibroblast. The one step growth curve of the rQUGFP was found to be identical with that of parent QU virus and the TCID50 titers of different generation recombinants maintained stable. These findings suggest that the region including ORF1, ORF8 and ORF9 of QU virus genome is dispensable for virus replication, and the foreign gene inserted into virus genome can be efficiently and stably expressed. The work lays the foundation for further studies of developing this virus as a vector of recombinant vaccine.

Abstract:In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells(ESCs) conditioned medium(ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement(KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses(ICMs) cultured in the control group with medium supplanted with fetal bovine serum(FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells’ self-renewal.

Abstract:To study the effect of the osmotic stress in the microenvironment on the growth and metabolism of the encapsulated cells under aerobic condition, Osmo-sensitive yeast Y02724 and high-osmotic resistant yeast Hansel were used as models to explore the growth and metabolism state of the cells cultivated inalginate-chitosan-alginate(ACA) microcapsules. The changes of the yeast cells’ specific growth rate, maximum product quantity and the secretion of ethanol and glycerol were analyzed. For Y02724, the yield of ethanol was increased in the ACA microenvironment compared to suspension cultivation. For Hansel, the maximum growth speed of microencapsulated cultivation had no obvious difference compared to the suspension cultivation. Moreover, after encapsulation, the production of glycerol was decreased for both Y02724 and Hansel compared to suspension cultivation. In conclusion, osmotic stress existed in the ACA microcapsules and affected the growth and metabolism of the cells.

Abstract:The low recovery of pertussis toxin (PT) and the low resolving efficiency of chromatography, due to the instability of PT in low salt condition, are the main challenges for its purification. We aplied 2 mol/L urea to prevent the aggregation and disassociation of PT during the purification by ion-exchange chromatography (IEC) and gel filtration chromatography (GFC). The effect of urea on the purification of PT was studied by ELISA assay and non-reduced SDS-PAGE. The activity recoveries of PT and filamentous hemagglutinin (FHA) in IEC and GFC, the resolution efficiency in GFC and the purities of PT and FHA were improved obviously by adding 2 mol/L urea in the mobile phase. The results highlight the potential application of urea in the acellular pertussis vaccine (APV) manufacture procedure.

Abstract:Phaffia rhodozyma is one of the organisms for production of astaxanthin, and the key process for extracting intracellular astaxanthin is cell disruption. In this work, cell disruption for extracting astaxanthin from Phaffia rhodozyma was studied with autoclave method at low acid concentration. The optimum disrupting conditions were: autoclave pressure 0.1 MPa, 121oC; hydrochloric acid concentration 0.5 mol/L; liquid to material ratio (V/W) 30 mL/g dry cell weight and disruption time 2 min. Under the optimum conditions, medium scale experiment showed that astaxanthin and total carotenoids recovery from Phaffia rhodozyma were (84.8 ± 3.2)% and (93.3 ± 2)%, respectively. This new method can lead to no poisonous residues and get high extraction yield, which have good prospects to be put into industrial production

Abstract:Monoclonal antibodies(McAbs) against human liver DnaJ-like protein(HLJ1) was produced by using lymphocyte- hybridoma technique and then one method for the detection of HLJ1 antigen was established. Two hybridoma cell lines which stably secreted monoclonal antibodies against HLJ1 were generated and named for A4C7 and C4C8. Subtypes of the two McAbs were both IgG1, and the antibodies showed high titer and good specificity. Using the prepared monoclonal antibody, human embryonic liver tissues were examined by immunohistochemistry. The results indicated that HLJ1 located in the cytoplasm of the human embryonic liver cell. A double antibodies sandwich ELISA was established by using C4C8 and HRP labeled A4C7. This assay had good specificity, and the lowest detection limit was 7.5 ng/mL and the linear range was 7.5~750 ng/mL. In conclusion, an immunohistochemistry method and a sensitive sandwich ELISA were established for the detection of HLJ1 protein.

Abstract:To screen the candidate genes associated with Musca domestica antibacterial peptides using DNA microarray technique, the hybrid probes were designed from the conservative domains of the encoded area of the insect antibacterial peptide genes in GenBank with biology software Designer 2.0, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter, those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by Escherichia coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analyzed using the software of MIDAS, fifteen valid hybridization signals were detected through two times of hybridization and scanning(the positive samples as a control were excluded). DNA microarray technique can be successfully applied screen the candidate genes associated with Musca domestica antibacterial peptides, and further provide significant evidence to discover its antibacterial peptide new genes.

Abstract:To investigate the effects of induction temperature on the expression product and the impact of urea concentration on the purification, HIV-1 Gag inclusion bodies from E. coli induced at 30oC (IB30) and 37oC (IB37) were dissolved with urea of different concentrations. The solubility and yield of refolding were compared. IB30 were dissolved with 2 mol/L and 8 mol/L urea, and then purified with chromatography. IB30 were found easier to be solubilized in low concentration of urea and easier to be refolded than IB37. Furthermore, compared to the IB30 dissolved in 8 mol/L urea, Gag protein solubilized in 2 mol/L urea was purified to higher purity with gel filtration (GF) and ion exchange (IEX) chromatography. Gag inclusion body induced at lower temperature may contain more protein with native-like or reversibly-denatured structures, and solubilization in the presence of low concentrations of urea can help to retain these structures. This study has provided new insights into the purification of proteins from inclusion bodies.

Abstract:A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker’s downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography(IMAC) . This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA(without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.