Expression and characterization of β-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-β-D glucosamine production / 生物工程学报

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Effect of glutathione on autologous fat graft survival of the rabbits / 中华整形外科杂志

Objective@#To investigate the effect of adding glutathion(GSH) to tumescent solution on autologous fat graft survival.@*Methods@#14 male and female New Zealand rabbits were divided into experimental group and control group randomly, 7 in each group. Experimental group: The donor areas of the rabbits were injected with 3 ml of tumescent solution with GSH. Control group: The donor areas of the rabbits were injected with 3 ml regular tumescent solution. DCFH-DA probe was used for fluorescent staining of harvested fat cells. Then stained fat cells were measured for the intracellular reactive oxygen species(ROS)content by fluorescence microplate. The grafts were harvested at 3 months after transplantation and assessed by general observation, volume measurement, wet weight measurement, HE staining for the number of fat cells, and CD34 immunohistochemical staining for the measurement of micro-vascular density. T test was performed by using SPSS 24.0.@*Results@#The intracellular ROS content of harvested fat cells in experimental group was lower than that in control group, and the difference was statistically significant (P<0.05). At 3 months after transplantation, the wet weight of fat grafts in experimental group was (1133.21±87.97) mg and that in control group was (718.79±79.27) mg. The volume of fat grafts in experimental group was (1.00±0.04) ml and that in control group was (0.59±0.03) ml. The number of fat cells in experimental group was (746.6±15.7)/10 high magnification vision and that in control group was (350.1±32.4)/10 high magnification vision. The density in group experimental was (8.1±2.0)/high magnification vision and that in control group was (6.7±2.4)/high magnification vision. The grafts′ volume, wet weight, and the number of fat cells in experimental group were higher than those in control group, and the differences were statistically significant (P<0.05). The difference of micro-vascular density between experimental group and control group had no statistical significance (P>0.05).@*Conclusions@#The addition of GSH to tumescent solution optimizes the process of autologous fat harvest, thereby improving the survival of fat graft.

Enhanced fat graft survival via sustained delivery of FGF-2 based on chitosan core-shell microspheres / 中华整形外科杂志

Objective@#To construct a novel carrier with core-shell structure-inner core of pFGF2-EGFP-loaded TACS coated by hydroxybutyl chitosan (HBC), and to explore its effects on granular fat graft survival.@*Methods@#The core-structured particles (TACS-pFGF2-EGFP) and core-shell-structured particles (HBC@ TACS-pFGF2-EGFP) were prepared to explore the release pattern of pFGF2-EGFP of these particles. The expression of FGF2 protein was detected by Western-Blot in 293T cells transfected with the sustained - release microspheres in vitro. Cell proliferation assay demonstrated that 10μg/ml pFGF2 plasmid could promote 293T cells growth. Eighteen New Zealand white rabbits were used for adipose tissue transplantation experiment. Rabbit left ear was treated as experimental group, 2 ml fat granules and HBC@TACS-pFGF2-EGFP were implanted; rabbit right ear was used as control group, 2 ml fat granules and HBC@TACS-empty plasmids were transplanted. The specimens were harvested at 4, 8, 12 weeks separately after fat transplantation. Gross view, HE staining, and immunohistochemical staining were performed to observe graft survival, biological characteristics, and neovascular density.@*Results@#HBC@TACS-pFGF2-EGFP particles could sustained release Pfgf2 gene in vitro, and successfully express FGF2 protein after transfecting 293T cells. At different time points after transplantation, the volume of adipose tissues was gradually reduced with time. The fat volume and survival rate of adipose tissues in the experimental group were significantly higher than that in the control group (P<0.05). HE staining showed that the arrangement of new adipocytes in the experimental group was more regular than that in the control group. Immunohistochemistry staining showed that the micro vessel density and FGF2 protein expression level in the adipose tissue of the experimental group were higher than those in the control group (P< 0.05).@*Conclusions@#HBC@TACS-pFGF2-EGFP particles with sustained release of FGF2 can improve the survival of granule fat transplantation.

Expression and characterization of a bispecific antibody targeting TNF-α and ED-B containing fibronectin / 生物工程学报

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.

Construction of directional T vector for gene cloning and expression / 生物工程学报

Traditional T vector cloning method requires onerous procedures for identifying recombinant, and directional cloning was impossible. In order to overcome these problems, we have devised a directional T vector pETG based on pET-23a(+). For gene cloning, 7 bp partial LacO sequence was introduced into DNA fragment to reconstitute a full length LacO with Bfu I digested T vector. After transformation, blue colonies were selected on LB plate supplemented with X-gal. Restriction enzyme digestion and PCR identification showed that all blue colonies contained the directionally inserted recombinants and the recombinant efficiency was nearly 100%. We have successfully cloned 103 genes from human liver cDNA; in the study complicated procedures for screening of recombinant were not required. Eight pETG clones were picked for protein expression, and all the clones successfully produced corresponding proteins. We demonstrated that the directional T vector was successfully constructed, and it was very suitable for gene cloning and expression.