Abstract:Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB.Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

Abstract:A strain Mortierella isabellinaM6-22-4,which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N'-Nitro-N-nitrsoguanidine(MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl₂ treatment. Transformation frequencies of 1.6～2.8 transformants/μg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1～2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.

Abstract:AIM To repopulate the liver of mice with acute liver injury and to make mouse models with chimeric liver by using human umbilical cord blood (hUCB)-derived mononuclear cells. METHODS Fifteen acute liver injury mouse models were induced by carbon tetrachloride intraperitoneal injection followed by two-thirds hepatectomy and all mice were divided into three groups: cell transplantation group (n=7), negative control group (n=3) and blank control group(n=5). HUCB cell preparations were transplanted into mouse spleens of cell transplantation group and phosphate bufferd saline (PBS) was injected into spleens of negative controls. Neither cell suspension nor PBS was given to the blank controls. Pathological changes were observed 7, 14 and 21 days after cell transplantation. Human albumin (ALB) and cytokeratin 19 (CK19) were also detected in the mouse sera and liver tissues. RESULTS All mice showed histological features of acute liver injury. Positive expression of human ALB and CK19 were observed in liver tissues of cell transplantation group 7, 14 and 21 days after cell transplantation. Human ALB could be detected from the sera and liver homogenates of cell-transplanted mice. No positive expression of human ALB and CK19 were observed in liver tissues and no human ALB was detected in sera of negative control group. CONCLUSIONS HUCB-derived mononuclear cells can differentiate into functional human hepatocytes and biliary cells in large quantity in mouse models with acute liver injury, thus a great progress were made in establishing mouse models with chimeric liver.

Abstract:A simple method of trypsin /collagenase I alternative digestion and iterative culture flask adherence to discard fibroblasts for bovine mammary cell culture was established in this study. By immunohistochemistry, flow cytometry, western blot, Electron microscopy analysis, the characteristics of bovine mammary cells were investigated in vitro. Effect of hyperthermia on the cell ultrastructures was also observed. The results showed that the mammary cells were diploid epithelia with intact 30 pairs chromatins, which could secrete alpha-casein into the medium .After exposed to hyperthermia, the cell condensed chromatin like crescent on the nuclei verges, mitochondria occurred expansion and vacuolization, and apoptotic bodies appeared, which suggested that heat stress could induce apoptosis of the mammary epithelia.

Abstract:According to the reported gene sequence of Rhizopus oryzae glucoamylases, the glucoamylase gene containing four introns was cloned from the total DNA of the natural Rhizopus arrhizu. Specific primers were designed to delete introns by overlapping PCR and a new cDNA sequence of Rhizopus arrhizu glucoamylase was obtained. The accession number in gene bank is DQ903853. This gene is successfully expressed in the Picha pastoris, producing a new protein with a high activity of glucoamylase.

Abstract:Methylotrophic yeast, Pichia pastoris was used to express recombinant batroxobin, and a technology route of producing recombinant protein was finally established. We synthesized batroxobin gene artificially by means of recursive PCR. pPIC9-batroxobin was constructed and transformed into Pichia pastoris GS115 (his4). Recombinant batroxobin was expressed in yeast engineering strain and it was purified from the culture supernatant. 10mg of recombinant batroxobin was purified from 1 liter fermentation media, it exhibited specific activity of 238 NIH units/mg and had molecular weight of 30.55 kD. The purified recombinant protein converted fibrinogen into fibrin clot in vitro, and shortened bleeding time in vivo. This study laid a foundation of development of hemostatic of recombinant snake venom thrombin-like enzyme.

Abstract:The argE gene from Escherichia coli coding for N-acety-L-ornithine deacetylase（NAOase）, the key enzyme involved in the L-arginine biosynthesis, had been cloned in pET22b and transformed into BL21(DE3). With 32.5% expression level in the optimal fermentation medium at 37℃, most NAOase was expressed as inclusion bodies. The soluble and active proportion could be slightly increased when expressed at low temperature. The specific activity of soluble NAOase purified by Ni-NTA resin chromatography was 1193.2u/mg. The species and proportions of whole cell proteins varied with induction conditions. The inclusion bodies expressed at 37℃ was more pure than 22℃after gradient wash with urea. Inclusion bodies could be partly refolding and reactivated by dilution and dialysis. Low protein concentration and suitable rate of oxidant/reducing agents were important to renaturation. In the optimal conditions 17.78% of Urea-denatured NAOase could be refolding and reactivated by dilution. The purified fusion protein was obtained after wash, solubilization and Ni-NTA resin affinity chromatography purification of inclusion bodies.

Abstract:To investigate the effect of Protein kinase B on the expression and location of p21 in mouse early development. Immunopreciptation technology was used to detect the localization of p21 and Western blotting was used to analyze the expression of p21 after microinjecting mRNA of WT-PKB,myt-PKB and PKB-KD to mouse eggs. There was no obvious difference between the three kinds of mRNA in the expression of p21. But the cell localization altered. The p21 retain in cytoplasm after microjecting myt-PKB. In mouse fertilized egg PKB/Akt controls the cell cycle by changing the cell localization of p21.

Abstract:Effect of CTAB addition on the accumulation of microbial transglutaminase(MTG) with Streptomyces hygroscopicus was investigated. The results showed that the addition of CTAB enhanced MTG accumulation, and the optimal addition time and concentration of CTAB were 32 h and 1%. The maximum MTG activity in the culture broth was 5.04u/mL and increased by 21.8% compared with the control. With the addition of CTAB, pro-MTG was activated to become MTG. CTAB could enhance the production of pro-MTG by relieving the product inhibition, and the accumulation of MTG was improved.

Abstract:Transplantation of the microencapsulated recombinant cells is a novel alternative approach to gene therapy of tumors. The semi-permeable membrane of microcapsule protects cells from host's immune rejection, increases the efficiency of gene transfer and reduces the need for frequent injection. Optimization of the preparation and culture is needed to acquire biological microcapsule with high cell viability and protein production. In this work, we studied the effect of different preparation and culture condition on the microencapsulated recombinant CHO cells growth and endostatin production. The result showed that the inoculum cells growth phase and seeding density potently affected the growth and endostatin production of the recombinant CHO cells in the microcapsule. The exponential growth phase recombinant CHO cells with a seeding density of 1×10⁶～2×10⁶ cells/mL microcapsules benefited to the cells growth and endostatin production. The time of preparation was another important effect factor of cells viability, the cells viability decreased with the increase of preparation time and the time of preparation should be under 5h for maintaining the cell viability and endostain production. The highest viable cell density and endostatin production was acquired when the microcapsule percentage was 5% in the culture of the microencapsulated cells, the cell growth and endostatin production decreased with the increase of the microcapsule percentage.

Abstract:To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) efficiently in pilot scale, cells of rhNDPK-A producing E. coli were homogenized by high pressure under 4℃, 950 Pa. The insoluble debris was removed by microfiltration and the soluble portion was concentrated by ultrafiltration. The resulted crude sample was loaded on DEAE-sepharose Fast Flow. The target fraction was collected and then load on Cibacron Blue 3GA Sepharose CL-4B. Eluted with buffer containing ATP from the AC column, rhNDPK-A was polished with ultrafiltration. The results showed that after homogenized 2 rounds, 1500g cells of E. coli brought crude sample containing 47.6g NDPK-A. Treated with microfiltration and ultrafiltration, 27.3g of NDPK-A were recovered from this bacteria homogenate. After 2-step purification with column chromatography and then polished with ultrafiltration, 17.2 g rhNDPK-A were collected with purity of 96.3%. The recovery of the whole purification process was 36.2%, and the productivity of rhNDPK-A was 1.15 g per 100 g wet cells. Comparing the recovery of each purification step, it was found that the recovery of polish is higher than that of affinity chromatography, which is higher than that of ion exchange chromatography. The limit step was the process of sample pretreatment among the 4 purification steps. Combine with the fermentation results reported before, it was deduced that the productivity of rhNDPK-A was 510 mg/L. In conclusion, an easily controlled purification condition with high yield provides material for the translation researches of NDPK; In addition, it was suggested the crucial step determine the recovery of non-secretive recombinant proteins might be the process of sample pretreatment, not be the process of column chromatography.

Abstract:The principal component analysis(PCA) was applied to the data processing in training sets, the new principal components were then used as input data for support vector machine model. A prediction model for optimum pH of chitinase was established based on uniform design. When The regularized constant C, epsilon and Gamma were 10, 0.7 and 0.5 respectively, the calculated pHs fitted the reported optimum pHs of chitinase very well and the MAPEs (Mean Absolute Percent Error) was 3.76%. At the same time, the predicted pHs fitted the reported optimum pHs well and the MAE (Mean Absolute Error) was 0.42 pH unit. It was superior in fittings and predictions compared to the model based on back propagation(BP) neural network.

Abstract:Aminolevulinic acid (ALA) is biosynthesized by the enzyme ALA synthase (ALAS). The ALA production has been studied using the overproducing ALAS from several bacteria in Escherchia coil, respectively. However, ALAS from eucaryote expressed in E.coli for producing ALA in the culture is not known. The extracellular ALA production and cell growth were investageted respectively using the recombinant E. coli overproducing Saccharomyces cerevisiae ALAS in shake-flask fermentations. The ALAS activity from the cell extract was assayed. The extracellular ALA was purified by the national-made large-dimension resins and confirmed by the capillary electrophoresis measurements. At 12h after induction at 37℃, the extracellular ALA production was up to 162mg per liter LB culture at initial pH 6.5 with exogenous levulinate, succinate and and glycine at the concentration of 20mmol/L respectively. The purity of ALA after purification is up to 90%.

Abstract:Acetyl-N-glucosaminyltransferase gene (nodC) was successfully cloned to Escherichia colifrom Mesorhizobium loti. The recombinant E.coli harboring nodC gene was able to synthesize chitooligosaccharides (COs) in MMYNG medium. In optimized condition, a yield of 526 mg/L was obtained after 26 h cultivation in 10 L bioreactor. COs concentration reached up to 4.5% of the cell dry weight. The COs products were purified by charcoal adsorption and Bio-gel P4 chromatography, penta-N-acetylchitopentaose （m/z, 1034［M+H］⁺）and tetra-N-acetylchitopentaose （m/z, 831［M+H］⁺） were identified as the dominating COs product using the method of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS).

Abstract:In order to build a protein expression system in a cold-adapted bacteriumAcinetobacter sp. DWC6, a promoter probe vector was constructed based on the plasmid pBR322. A fragment containing the promoter of the β-lactamase gene (the ampicillin resistance gene) in pBR322 was eliminated and replaced by a fragment comprizing a kanamycin resistance gene amplified from pJRD215. DNA fragment harboring in the Acinetobacter species specific ori was also inserted into the plasmid pBR322 to construct a promoter probe vector named pBAP1, which could replicate both in E. coli and in Acinetobacter sp. DWC6. The promoter selection library was constructed by randomly inserting genomic DNA fragment of Acinetobacter sp. DWC6 at upstream of reported gene, and target promoters were screened from genomic library on ampicillin selection plates. The function of pBAP1 and isolated promoters were determined by detection of the ampicillin sensitivity and the expression level of β-lactamase in the host cell.

Abstract:The efficiency of the exogenous DNA transfecting mouse sperm was studied by the DIG end labeled and immunohistochemistry technology. The results suggested that: the efficiency of transfecting positive rate of individual mouse sperm was distinct difference (P<0.01), and the average rate was 13%. The acrosomal reaction was evaluated using the technology of Coomassie brilliant blue stained, and the appropriate in vitro fertilization (IVF) medium TYH was elected. Mouse sperms were transferred with GFP gene in vitro, and the mature oocytes were fertilized using IVF, and then the zygotes were cultured in vitro. The embryos were observed using the fluorescence microscopy, and the transgenic rate was 4.7%. The results suggested that sperm mediated gene transfer (SMGT) was an effective and feasible method.

Abstract:Entire 3ABC sequence of FMDV containing a 6×his tag coding sequence at the N-terminal was obtained through PCR amplification using a pair of specific primers, subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence and finally constructed recombinant plasmid of pMel-3ABC. After co-transfected the recombinant plasmid and linearized Bac-N-Blue^TM DNA into Sf9 insect cell under intermediary agent of the Cellfectin^?, the result showed that we have already acquired recombinant baculovirus by screen of plaque assay and identification of PCR. Though the recombinant baculovirus infecting the Sf9 cells again, experiments indicated that 3ABC gene could express in insect cells and the expressed protein was secreted in the supernatant of Sf9 cell culture possessing favourable biological activities detected by adopting two methods of SDS-PAGE and Western blot. The result verified that the protein could respond with sera derived from FMDV infected animals, but have no responsibility with sera derived from health animals and vaccinated animals detected by indirect ELISA using antigen of expressed protein after purification with Ni-NTA his bind resin. Therefore, this study has established a solid foundation for establishing an effective diagnosis method to discriminating the FMDV infected animals from vaccinated animals.

Abstract:Recently many reports have described that recombinant baculovirus could serve as a new gene transfer vehicle for mammalian cells with many unique advantages. In this study, the constructed recombinant baculovirus BacV-CMV-EGFPA containing the enhanced green fluorescent protein (eGFP) gene driven by CMV promoter was used to explore the feasibility of improving the efficiencies of transduction experiment in CV-1 cells by centrifugal method. Refer to the centrifugal transduction protocol of recombinant lentivirus, CV-1 cells were incubated with the culture supernatant of Sf-21 cells infected by BacV-CMV-EGFPA (moi=30) and then centrifuged at 600g for 1h at RT, reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM) 48h post transduction. Results showed that centrifugal method can achieve higher gene delivery and expression efficiencies than transduction by simple virus-cell mixing for 4h at 27℃ with least impairment to cell viability. The centrifugal transduction protocol was further optimized by testing different centrifugal times, post-centrifugation incubation times and surrounding solutions. We found that centrifugation at 600g for 1h at RT is sufficient to achieve the highest transduction efficiencies in target cells and PBS is more suitable than other surrounding solutions. Compared with previous protocol in which tranduction occurs for 4～8h at 27℃, centrifugal method developed in this study could achieve more higher transduction efficiencies in more shorter time. Nine different mammalian cell lines (CV-1, 293FT, HepG2, 293T, CHO, C127, MT4, H9, Molt-4) were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the BacV-CMV-EGFPA supernatant (moi=30) at 600g for 1h in PBS surrounding solution at RT. Results showed that most mammalian cell lines used in this study could be effectively transduced with recombinant baculoviruses by centrifugal method, and more higher and satisfactory transduction efficiencies could be achieved in primate adherent culture cells than in suspended culture cells. These results show that the baculovirus centrifugal transduction protocol have notable advantages: more rapid, efficient and nontoxic, and could be easily used in daily common experiments.

Abstract:Hypodermin C( HC) cDNA was amplified from recombinant pGEM－T/HC， cloned in frame with the signal sequence in yeast vector pPIC9k. The plasmid was linerarized and transformed into Pichia pastoris GS115 strain by electroporation method. Recombinant strain was screened by G418 resistant, and further confirmed by PCR. The recombinant strain which contains insert was induced in the medium containing 0.5% methanol. The supernatant was collected and then purified by anion exchange chromatography. SDS-PAGE indicated that the target protein is around 28kD. Western-blot showed it can react with rabbit-anti HC serum. Gelatin substrate SDS-PAGE displayed it had enzyme activity. Provided a method to produce enough antigens for carrying out extensive immunological analyses.