Abstract:Cyclodextrin glucanotransferase, the essential enzyme for the production of cyclodextrins, has become the focus of scientific research nowadays. Although many related enzyme properties are well known, the crucial factors in product specificity determination remain to be answered. Here, the recent research progresses of cyclodextrin glucanotransferase, especially those about the evolution of product specificity, were reviewed, and the scientific problems were discussed.

Abstract:Collagen is the most abundant protein in human body and a periodic helix, i.e., triple helix, fibrous protein, which provides the scaffold structures for the cell adhesion and macromolecule aggregation, etc. With the development of gene engineering and biomaterial technologies, and the incessant studies on the technique to obtain the proteins with special functions, the collagen protein has been one of the third generation biomaterials that attract more attention than others. In this paper, we reviewed the recent structure-based design and biosynthesis of collagen.

Abstract:Adipose tissue contains a population of multipotent cells called adipose-derived stem cells (ADSCs). With the similar properties of marrow-derived mesenchymal stem cells, ADSCs have the ability to differentiate differentiate towards adipogenic, osteogenic, chondrogenic, myogenic, endothelial, hematopoietic, hepatic, islet, and neurogenic cell lineages. As adipose tissue in harvested in large amounts with minimal morbidity, it can be widely used in tissue engineering, organ repair and gene therapy. This paper focused on the plasticity of ADSCs and reviewed the new advances of this field. Finally, the problems and prospect for application was also discussed.

Abstract:1,3-propanediol production by microbial fermentation has become the research hot spot for its amiability with the environment. Here the molecular mechanism of glycerol bioconversion to 1,3-propanediol was outlined by elucidating the fermentation strains, metabolic pathways, regulon and key enzymes. Of enzymes, glycerol dehydrogenase, the velocity-limiting enzyme in glycerol reductive pathway, was emphatically discussed with regard to its molecular structure and reactivating factors. This paper aims to provide the basis for genetic modification of fermentation strains.

Abstract:S-adenosylmethionine-dependent uroporphyrinogen Ⅲ methyltransferase (SUMT) is a novel red fluorescence indicator. However, the production of SUMT in Escherichia coli is restricted by its relatively low solubility, and little is known about the red fluorescent materials that are associate with SUMT. Two individual SUMT mutations, L166A and L88R/L89G double mutant were produced by site-directed mutagenesis. Both mutants were overexpressed in E. coli and purified by Ni-NTA chromatography. The reddish mixtures isolated from the purified L88R/L89G double mutant were analyzed by UV-visible spectra scanning and mass analysis(MS). The L88R/L89G double mutant has enzymatic activity in vivo, whereas L166A mutant loses the activity. Trimethylpyrrocorphin is identified as the main constituent in the isolated pigments. The purified L88R/L89G mutant increases protein solubility, which is applied potentially as the fluorescent indicator denoting the solubility of protein fusion partner.

Abstract:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important pathogen. One of the important virulence traits of EHEC O157:H7 is the capacity to produce attaching and effacing (A/E) lesions on enterocyte. This property encoded by a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE contains ler (LEE-encoded regulator) gene. The product of ler is a central up-regulator of many virulence genes of the LEE. Another important virulence factor of EHEC O157:H7 is Shiga toxin (Stx), encoded by a prophage integrated into the chromosome of O157:H7. In order to obtain an attenuated vaccine candidate, a ler deletion mutant of O157:H7 was constructed by use of suicide vector pCVD442. Meanwhile, due to potential instability of the prophage carrying the stx gene, the prophage was cured with serial passages of bacteria and confirmed by PCR and DNA sequencing. A ler/stx deletion mutant of EHEC O157:H7 was constructed, termed as O157:H7(Δler/Δstx). The cultural supernatant of O157 ler/stx deletion mutant was inoculated in vero cell culture, and the result indicating that O157 ler/stx deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the O157 ler/stx deletion mutant and the virulent strain O157:H7 EDL933, respectively. Mice were observed daily for clinical signs and weight changes. After inoculation of the deletion mutant, test group of mice (inoculated with O157:H7(Δler/Δstx)) gained weight normally and experienced no clinical signs. In contrast, control group of mice (inoculated with O157:H7) exhibited weight loss and all died in four days. In another experiment, pregnant mice were orally vaccinated by O157:H7(Δler/Δstx) twice at interval of 14 days. Subsequently, the suckling mice were orally challenged with O157:H7 EDL933 at 7 days of age. The results showed that 78.34% of the sucking mice born by vaccinated mice were survival and 12.73% of the sucking mice born by non-vaccinated mice were survival. This study demonstrated that O157 ler/stx deletion mutant lost the toxigenicity to vero cell and to be safety to mice. Oral immunization can induce specific immune responses, and this mutant strain could be used as an attenuated vaccine candidate against EHEC O157.

Abstract:To prepare a novel fusion protein (tTF/SA) as a universal effector for targeting therapy of blood coagulation and to analyze its biological activities. The fusion gene tTF/SA was constructed by PCR, then inserted into expression vector pET22 b (+)，and expressed in E.coli BL21 (DE₃). The fusion protein was purified using Nickel-affinity chromatography column. The activities of tTF moiety of the fusion protein were analyzed by clotting assay and FX activation assay, and the binding activities of Streptavidin(SA) to Biotin(B) were analyzed using ELISA. Results: The recombinant plasmid tTF/SA/pET22 b(+) with the correct sequence was obtained. The fusion gene tTF/SA was expressed with high yield in E.coli BL21(DE₃). The purified fusion protein retain the abilities of activating FX, inducing blood coagulation, and binding Biotin. The fusion gene tTF/SA was successfully expressed in E.coli BL21(DE₃). The recombinant tTF/SA proteins retain the activities of TF and SA. The multitarget therapy of selectively inducing thrombosis in tumor blood vessels can be achieved by the combination of tTF/SA, as a universal effector, and biotinlated carriers directing to tumor blood vessels.

Abstract:To generate transgenic porcine which expresses human serum albumin（HSA）, the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2kb 5^′ regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron.The resistant gene neo was inserted into intron 1 and tk was ligated to the 3^′ end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

Abstract:This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI(10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100～200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI(10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.

Abstract:This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI(10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100～200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI(10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.

Abstract:This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI(10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100～200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI(10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.

Abstract:This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI(10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100～200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI(10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.

Abstract:Recently, mesenchymal stem cells (MSCs) have been one of the target cells of gene engineering. To construct the lentiviral (LV) vectors carrying the brain-derived neurotrophic factor (Bdnf) gene, the rat mesenchymal stem cells (rMSCs) were infected and finally the Bdnf gene-modified rMSCs was obtained. The CDS region of the rat Bdnf gene was obtained with reverse transcriptase-polymerase chain reaction (RT-PCR), and the transfer plasmid (PNL-BDNF-IRES₂-EGFP) of the LV vector was constructed. The three plasmids of LV vector: PNL-BDNF-IRES₂-EGFP, HELPER, and VSVG were cotransfected to 293T cells to produce the LV vectors, which enabled the coexpression of the Bdnf gene and the enhanced green fluorescent protein (Egfp) gene. rMSCs were separated from the bone marrow of 2-month-old F344 rats, cultured in vitro, and identified. rMSCs were infected by the LV vectors that were produced already and were identified with fluorescent microscope, RT-PCR, immunocytochemical staining, and western blot. The result of sequencing showed that the sequence of the cloned Bdnf gene was consistent with that reported in the GenBank. The PNL-BDNF-IRES2-EGFP plasmid that was identified showed the correct sequence. After the 3 plasmids of LV vectors were cotransfected to the 293T cells, considerable green fluorescence in 293T cells was observed under the fluorescent microscope; the supernatant was collected and concentrated using ultracentrifugation, and the titer of the replication_defective LV vector particles measured was found to be 6.7×10⁷ TU/mL. After the constructed LV vectors infected the rMSCs, the results obtained using RT-PCR, immunocytochemical staining, and western blot showed that the expression of BDNF in the Bdnf-rMSCs group (experimental group, EG) was significantly higher than that in the PNL-IRES2-EGFP-rMSCs group (mock group, MG) and the rMSCs group (control group, CG) at both mRNA and protein levels. LV vectors carrying the Bdnf> gene were constructed successfully. The Bdnf gene-modified rMSCs could express BDNF to a higher degree. This greatly facilitates the next step in the study, such as the long period of therapeutic observation of cerebral ischemia with Bdnf gene-modified rMSCs.

Abstract:Based on the sequence of BAC (Bacterial Artificial Chromosome) along with the Cre/lox P system, the gene-targeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed. The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by NotⅠ/HindⅢ and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BAC-TDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene_targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of Ⅰ-SceⅠ and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100～300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.

Abstract:The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase Ⅳ digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10⁶～10⁸ cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self_renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

Abstract:Midbrain dopamine (DA) neurons play an essential role in modulating motor control. Defects in central DA neurons affect a wide range of neurological disorders including Parkinson's disease (PD). The greatest motivation in the field has been the potential use of DA neurons for cell transplantation therapy in Parkinsonian patients. Recent studies indicated that BMSCs could differentiate into DA neurons in vitro as neural stem cells (NSC) and embryonic stem cells (ESC) could. However, there are no direct evidences about functional DA neurons derived from BMSCs. According to the protocols which had been applicated in inducing neuronal stem cells and embryonic stem cells differentiate into DA neurons in vitro, the present study provides a protocol by using 50 μmol/L brain derived neurotrophy factor (BDNF), 10 μmol/L forskolin (FSK) and 10 μmol/L dopamine (DA) to induce BMSCs differentiate into DA neurons. After 2 weeks of differentiation, the cells expressed the character of neurons in ultrastructure. RT-PCR discovered mRNA of NSE(neuron specific enolase), Nurr1, Ptx3, Lmx1b and Tyrosine hydroxylase (TH) were positive. Immunocytochemistry staining indicated the ratio of TH-positive neural cells was significantly increased after induced 2 weeks(24.80±3.36)% compared to that of induction of 3 days(3.77±1.77)%. And the DA release was also different between differentiated and undifferentiated cells detected by high performance liquid chromatography(HPLC). That is to say BDNF and FSK and DA can induce BMSCs differentiate into DA neurons in vitro, and the transdifferentiated cells express mature neurons characters. BMSCs might be a suitable and available source for the in vitro derivation of DA neurons and cell transplantation therapy in some central neural system diseases such as PD.

Abstract:In the present study, antioxidant activities of the fruits of A. heterophyllus, A. squamosa, T. bellirica, S. samarangense,A. carambola and O. europa were investigated. For this, at first matured fruits of them were sliced into small pieces and dried in the sun and finally crushed in a grinder to make powder. Ethanolic extracts of fruit powder were prepared using 99.99% ethanol. The antioxidative activities of these extracts were determined according to their abilities of scavenging 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical .It was demonstrated that all the ethanolic extracts of A. heterophyllus, A. squamosa, T. bellirica, S. samarangense,A. carambola and O. europashowed antioxidant activities. The IC50 of the ethanolic extracts of A. heterophyllus, A. squamosa, T. bellirica, S. samarangense,A. carambola and O. europa were 410, 250, 34, 200, 30 and 76μg/mL, respectively. Among them, A. carambola showed the highest antioxidant activities followed by T. bellirica, O. europa, S. samarangense, A. squamosa and A. heterophyllus indicating that fruits of A. carambola, T. bellirica and O. europa are very beneficial to human health.

Abstract:Nanohydroxyapatite/chitosan composite scaffolds were fabricated and the proliferation and differentiation of preosteoblast MC 3T3-E1 on them were examined for the assessment of their biocompatibility. Nanohydroxyapatite was combined with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 3T3-E1 cells were inoculated into the porous composite scaffolds and chitosan scaffolds, respectively. The morphology of cells cultured on the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTT assay. After being cultured in conditioned medium for 30 days, the cells' alkaline phosphatase activities on the scaffolds were studied in situ to compare their differentiation levelabout. Moreover, the alkaline phosphatase activities were assessed with a kit. The expression level of characteristic osteogenic gene was evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The results indicated that MC 3T3-E1 cells grown on the composite scaffolds showed a higher proliferation rate and spread better than that on chitosan scaffolds. The alkaline phosphatase stain results showed that the alkaline phosphatase activity of cells on composite scaffolds was significantly higher than that on the chitosan scaffolds. In addition, the quantitative examination of alkaline phosphatase activity indicated that the cells cultured on the composite scaffolds expressed an activity level about 8 times higher than that on chitosan scaffolds. Simultaneously, the osteogenic gene osteopontin (OPN) of cells cultured on composite scaffolds showed a higher expression level than that on chitosan scaffolds. Another osteogenic gene osteocalcin (OC) was expressed in cells cultured on composite scaffolds, whereas it was not detected in cells on chitosan scaffolds. The addition of nanohydroxyapatite in the scaffolds improved not only the proliferation but also the differentiation of preosteoblast cultured on them. The composite scaffolds showed good biocompatibility and bioactivity. These scaffolds would be promising in bone tissue engineering.

Abstract:The purification and the characteristics of an enzyme from Morganella morganii J-8, which could produce d-pseudoephedrine from 1-phenyl-2-methylamine-acetone, were performed in this study. In this research, first, cells were disrupted by ultrasonic treatment at 4℃. The carbonyl enantioselective reductase was purified with a combination of ammonium precipitation, Phenyl Superose hydrophobic chromatography, DEAE anion exchange, and native polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme subunit was estimated to be 42.5kD on sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). The native molecular mass of the enzyme that was analyzed by high-performance liquid chromatography was found out to be 84.1kD, which indicated that the enzyme was a dimmer. The purified enzyme was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the result showed that the purified enzyme had high homology with leucine dehydrogenase.

Abstract:Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A.fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5～7.0, but the pH stability of Q23L was lower than A.fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A.fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51u/mg of the wild type to 109u/mg at pH5.5. Meanwhile, the pH stability of Q23L decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0～4.5 and pH6.5～7.0 has been improved compared with Q23L.The specific activity of Q23LG272E basically maintained at the level of Q23L.Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E，and discussion for the relationship between structure and function of phytase was given.

Abstract:To obtain thermostable aspartate aminotransferase, the gene aspC from an extremely thermophilic bacterium, Thermus thermophilus HB8 was cloned, and its product was overexpressed in Escherichia coli BL21(DE3) and Rosetta(DE3). The expression in Rosetta(DE3) was more efficient. The optimum reactive pH was 7, and the recombinant enzyme activity changed little when incubated in the buffer of pH8～10 on 37℃ for 1 h. The optimum reactive temprature was 75℃, and the recombinant enzyme was more stable on the temperature of 25～55℃. The half life of recombinant enzyme on 65℃ was 3.5 h, on 75℃ was 2.5h. K_m^KG was 7.559mmol/L, V_max^KGwas 0.086 mmol/(L·min)， Ksub>m^Asp was 2.031mmol/L， V_max^Asp was 0.024mmol/(L·min). Ca²⁺，Fe³⁺，Mn²⁺ inhibited enzyme activity softly.

Abstract:HLA-A^*2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8⁺ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A^*2402 tetramer loaded with HCMV pp65_341-349 peptide (QYDPVAALF, QYD). The cDNA of HLA-A^*2402 heavy chain was cloned by RT-PCR from one of the donors．DNA fragment encoding the ectodomain of HLA-A^*2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A^*2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A^*2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A^*2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of β₂-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A^*2402-QYD monomers with streptavidin-PE at a molar ratio of 4∶1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24⁺ donors and the frequencies of tetramer-binding CTL were 0.09％～0.37％ within total CD8⁺ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A^*2402 individuals.

Abstract:Previously, an mAb 10F7 was developed against H5N1 hemagglutinin, which was highly specific to 34 different H5N1 strains and showed good neutralizing activity. In the present study, the single-chain fragment of the antibody was cloned into a prokaryotic vector and then expressed in E. coli. The activity of the scFv was tested in hemagglution inhibition and neutralization experiment. Two H5N1 virus strains were inhibited to bind erythrocyte cells by the scFv while the H9 virus was not. Also, five H5N1 virus strains were neutralized during infecting MDCK cells. These results showed an approachable method for developing therapeutic antibody to H5N1 virus.

Abstract:A marine unicellular green alga, Platymonas subcordiformis, was demonstrated to photobiologically produce hydrogen gas from seawater. The objective of this study was to localize and identify the hydrogenase isolated from P. subcordiformis. Adaptation in the presence of inhibitors of protein biosynthesis indicated that the hydrogenase was much more inhibited by cycloheximide than that by chloramphenicol. The result suggested that the hydrogenase isolated from P. subcordiformis is probably synthesized in cytoplasmic ribosomes. Both Western blot analysis and immunogold electron microscopy demonstrate that theP. subcordiformis hydrogenase is mainly located in the chloroplast stroma. The proteins that reacted specifically with the antibodies against the iron hydrogenase isolated from Chlamydomonas reinhardtii were concentrated by immunoprecipitation. The separated protein bands were cut out of the SDS-PAGE gel, in-gel digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Mascot was employed for analysis of the MALDI data using the public databases NCBInr. The hydrogenase isolated from P. subcordiformis was identified to be the Fe-hydrogenase.

Abstract:Microencapsulated recombinant cells technology is a novel approach to tumors therapy. It is necessary to prepare a plenty of the microcapsules with better cell viability and higher endostatin production in order to bring this technology into the clinic. The in vitro culture and cryopreservation are very important parameters in the preparation of microencapsulated cells. In this work, we studied the effect of the in vitro culture and cryopreservation on microencapsulated recombinant cells growth and endostatin production and the effect of the in vitro culture on the cryopreservation of microencapsulated recombinant cells. The results showed that the time of in vitro culture potently affected microencapsulated recombinant CHO cells growth in vitro, endostatin production and the microcapsule stability. The microcapsule kept intact after 36 days of implantation when thein vitro culture time was under 4 days. The thawed microencapsulated recombinant CHO cells had better cell growth and higher endostatin production after 40 days of cryopreservation when the in vitro culture time was 4 days and 8 days. Therefore, the best in vitro culture time was 4 days according to the results of the in vitro culture and cryopreservation and the cryopreservation did not affect microencapsulated recombinant CHO cells growth in vitro, endostatin production and the microcapsule stability.

Abstract:A dominant H-2^d restricted Th epitope P34 was found to be contained in recombinant particulate hepatitis E virus (HEV) vaccine HEV 239. In this paper, the cellular immune response induced in P34 immunized BALB/c mice were studied and the priming effect of P34 was characterized. Groups of BALB/c mice were subcutaneously (s.c.) immunized with P34, splenocytes were then stimulated with P34 and HEV 239 protein, cellular immune response was assayed by IFN-γ-ELISPOT, flow cytometry and T cell proliferation experiments. Results showed that P34 immunized BALB/c splenocytes responsed to P34 and HEV 239 protein stimulation in IFN-γ-ELISPOT, flow cytometry and T cell proliferation experiments. After depletion of the CD4⁺ T cells from the immunized splenocytes by magnetic separation, the response decreased to the background level while almost no influence was observed after CD8⁺ T cells depletion which showed that the cells responsible for IFN-γ secretion were mainly CD4⁺ T cells. Then mice were primed with P34 and boosted with its vector protein, E2, the E2 specific antibody titer were assayed. Results showed that after P34 priming, some of the 10μg, 20μg E2 boosted mice could develop anti-E2 antibody 1 week later and all the mice had detectable antibody 3 weeks after boosting. In the control peptide P18 priming group, even after boosting with 20μg E2, anti-E2 antibody couldn't be detected until the end of this experiment. The results showed that priming with P34 epitope could increase the immunogenicity of its vector protein, E2, in BALB/c mice.

Abstract:Lactobacillus casei 393 was selected as a bacterial carrier for the expression of Porcine Parvovirus（PPV）protective antigen VP2 protein. The gene encoding PPV VP2 protein was cloned into the Lactobacillus casei surface expression vector pPG, and then the constructed recombinant vector pPG-VP2 was electrotransformed into Lactobacillus casei 393 generating the recombinant system pPG-VP2/L.casei393 expressing PPV VP2 protein. The recombinant strain was induced by 2% Lactose in MRS and about 74kD protein was detected with SDS-PAGE. The result of Western-blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescent test showed that the expressed protein was secreted on the cell surface Lactobacillus casei.

Abstract:Canine adenovirus type 2 (CAV-2) has been proposed as a vector for recombinant vaccine. Alternatively, it may be an attractive tool for gene transfer due to lack of pre-existing immunity in humans. In this study, a transfer vector based on CAV-2, in which the 1381bp fragment of the E3 region was deleted, and a linker containing the NotⅠ,ClaⅠ,FseⅠ restriction enzyme sites were cloned into the deleted region. The recombinant CAV-2 genome was released from the plasmids enzyme digestion and transfected into MDCK cells by lipofectamine to obtain the recombinant virus. No significant difference in morphology, hemagglutination and replication between the recombinant and the wide type CAV-2 was found. These results indicated that this recombinant virus CAV-2-ΔE3（NF）may be an efficient vector for gene transfer and the capacity of the vector for inserted foreign gene was up to 3.3kb.

Abstract:With PVA as the carrier, the frozen beer yeast cells were immobilized for production of CTP from CMP. we explored the optimal condition of the immobilization from the aspects of the type, concentration of the PVA, and the immobilizing methods of cells In all 8 continuous batch of fermentation under the reactional condition of the immobilized cells, the conversion rate of CTP were maintained about 85%～95%. Moreever, the storage stability of immobilized cells were investigated, and the products was also isolated and identifided by HPLC.

Abstract:The effect of outer spermine on cell growth, accumulation of polysaccharides and utilization of nutrient together with the intracellular polyamine contents were investigated in suspension cultures of protocorm-like bodies from Dendrobium huoshanense. The results indicated that spermine at 0.6mmol/L was the most effective in increasing cell growth and polysaccharide synthesis. The specific growth rate of cell increased from 0.046d^-1 to 0.054d^-1, and the maximum dry weight and polysaccharide production reached 32.4g DW/L and 2.46g/L respectively, which were 1.32-fold and 1.31-fold that of the control on day 30.The titres of intracellular free polyamines were higher in the cultures treated with spermine than that of the control. Invertase and nitrate reductase activities were found to increase significantly in the cultured cells treated with spermine, which was beneficial to the utilization of carbon and nitrogen source.

Abstract:Metabolome has become an important part of Systems Biology, and a large set of data has already gained by applying the methods of metabolome. How to deal with the data and how to combine data of metabolome with data of other omics are problems that can not be ignored. An Enzyme Amount Multiple Factor was imported into the enzyme kinetic equation. When the enzyme amount in the system changed, in silico model, it means to alter the Enzyme Amount Multiple Factor. In order to observe ethanol concentration response to enzyme amount changes in S. cerevisiae glycolysis pathway model, enzyme amount was separately set at high and low level, the corresponding Enzyme Amount Multiple Factor value was 10 and 0.1, relatively. Based on the result of simulation, twelve enzymes in pathway were separated into two classes, classⅠand classⅡ by cluster analysis. The four enzymes belonging to classⅠ, ADH, HK, PFK and PDC, all catalyze irreversible reactions. The six out of eight enzymes belonging to classⅡ, ALD, GAPDH, GlcTrans, lpPEP, PGI and TIM, catalyze reversible reactions. The other two enzymes belonging to classⅡ, lpGlyc and PK, catalyze irreversible reactions. Based on this method, data of metabolome and proteomics are easily integrated to accomplish relatively overall analysis of system properties.

Abstract:This paper studied the effect of polyethylene glycol (PEG) on regeneration and free proline accumulation of callus of Pogonatherum paniceum (Lam.) Hack. under motionless liquid culture condition and shake liquid culture condition. Callus of P. paniceum had the ability to resist the stress of PEG. The effects of PEG stress and culture conditions on the callus of P. paniceumappeared mainly in two aspects, delaying regeneration time and debasing regeneration rates. The shake liquid culture mainly delayed the regeneration time and PEG stress mainly debased the regeneration rates. Free proline accumulated in the two culture conditions, and the contents of proline were positively correlated with PEG concentrations and culture time. After stress removal, most of the callus could recover the ability of regeneration, and the free proline might pay an important part in the inhibition and recovery. So it must be chosen a more than 300 g·L^-1 PEG concentration and long than 3 weeks culture time in the selection of drought-resistant mutants of P. paniceum. The motionless liquid culture was more suitable for selection of drought-resistant mutants.

Abstract:To establish a new high-throughput screening model for the agonist of PPARδ,PPARδ gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARδ fragment was excised by restriction enzymes, and inserted into pTARGET^TM Vector to construct expression vector pTARGET^TM-ppARδ. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE×3-luc. The vector pTARGET^TM-ppARδ was transiently cotransfected with pGl3-PPRE×3-luc into different cell lines to assay the expression levels of luciferase. The PPARδ agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARδ, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.

Abstract:In order to optimize the conditions of construction BAC library, the transformation efficiency of E.coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19×10¹⁰ cfu/μg pUC19 DNA) when harvesting the cells between an OD₅₅₀ of 0.7～0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.

Abstract:Secondary lymphoid-tissue chemokine (SLC) is a type of CC chemokine identified by searching the Expressed Sequence Tag (EST) database. The full-length SLC gene was synthesized based on human SLCsequence using SOE-PCR. The sequenced SLC gene was cloned into expression vector pTMF and pALM, which used to transform Escherichia coli. Then the E.coli was cultured and induced according to protocol. The expressed target protein was identified by Western blotting. The target protein was expressed as soluble protein as well as inclusion bodies, the ratio of these two forms target protein varied with the difference conditions of culture and induction. The target protein was purified with the methods of nickel-nitrilotriacetic acid(Ni-NTA) metal-affinity chromatography. The results of electrophoresis of the purified target protein showed that the molecular weight was larger than the predicted molecular weight.

Abstract:Secondary lymphoid-tissue chemokine (SLC) is a type of CC chemokine identified by searching the Expressed Sequence Tag (EST) database. The full-length SLC gene was synthesized based on human SLCsequence using SOE-PCR. The sequenced SLC gene was cloned into expression vector pTMF and pALM, which used to transform Escherichia coli. Then the E.coli was cultured and induced according to protocol. The expressed target protein was identified by Western blotting. The target protein was expressed as soluble protein as well as inclusion bodies, the ratio of these two forms target protein varied with the difference conditions of culture and induction. The target protein was purified with the methods of nickel-nitrilotriacetic acid(Ni-NTA) metal-affinity chromatography. The results of electrophoresis of the purified target protein showed that the molecular weight was larger than the predicted molecular weight.