Biomedical subjects

Ning Deng

Publications and source records attributed to Ning Deng.

10 recordsLinked to original sources

PubMed2006

[Expression of human anti-HBsAg single chain Fab gene in Pichia pastoris].

AIM: To investigate the relation of the affinity activity to the structure of recombinant Fab. METHODS: The human anti-HBsAg single chain Fab gene was obtained by overlaping PCR from the H and L chains of Fab, and was introduced into the expression system of Pichia pastoris. The expression vector of single chain Fab was transformed into GS115 cells by the method of LiCl transformation. The transformants were obtained and cultured in shake flask. The culture supernatant of the recombinant yeast was deposited in (NH(4))(2)SO(4) and purified by affinity chromatography. The affinity of culture supernatant of recombinant yeast and purified single chain Fab was analyzed by ELISA. RESULTS: The analysis of SDS-PAGE and Western blot showed that the single chain Fab gene was expressed successfully in Pichia pastoris, and the expression quantity was about 5-10 mg/L in shake flask. The recombinant Fab was purified by affinity chromatography and the purity reached 97.8%. The ELISA analysis showed that the recombinant single chain Fab had good HBsAg binding properties. CONCLUSION: The single chain Fab gene could be expressed successfully in Pichia pastoris and the recombinant single chain Fab can efficiently bind with HBsAg.

Electrophoresis, Polyacrylamide Gel↗

PubMed2005

Reversed-phase electrochromatography with a monolithic microcolumn prepared in a 2.2-mm-inner diameter fused-silica tube.

Capillary electrochromatography possesses advantages of high separation efficiency and velocity, but it also has its disadvantages due to its small inner diameter, such as poor detection sensitivity, low sample capacity, and some trouble in its column preparation. To overcome these shortcomings, a monolithic microcolumn with a surface area larger than 200 m2/g was prepared by sol-gel polycondensation of tetraethoxysilane-hydrochloric acid-poly(ethylene glycol) and filling with fine quartz sand in a 2.2-mm-i.d. fused-silica tube. The prepared microcolumn was used in the separation of aromatic compounds by reversed-phase electrochromatography. Some factors that affected electroosmotic flow were explored, such as electric field strength, buffer concentration, and buffer pH. Acetonitrile concentration in the mobile phase was investigated for phenol, benzene, and naphthalene separation. The separation results were satisfying with the electrochromatographic microcolumns. The detection limits of phenol, benzene, and naphthalene were 0.07, 0.26, and 0.04 mg/L, respectively.

Journal Article↗

PubMed2005

Characterization of the performance of optical amplitude-shift keying-differential phase-shift keying orthogonally modulated signals.

We analytically investigate the signal performance of orthogonal modulation with slow amplitude-shift keying (ASK) data superimposed upon a high-speed differential phase-shift keying (DPSK) signal. The receiver sensitivities for both the ASK and the demodulated DPSK signals are formulated based on a probability model, and their dependence on the bit rate and the extinction ratio of the ASK data is theoretically investigated. A simple result of analysis of DPSK data performance has been derived. We verified the analytical results by experimental measurements.

Journal Article↗

PubMed2005

Regulation of PTEN by Rho small GTPases.

PTEN (phosphatase and tensin homologue) is a phosphatase that dephosphorylates both protein and phosphoinositide substrates. It is mutated in a variety of human tumours and has important roles in a diverse range of biological processes, including cell migration and chemotaxis. PTEN's intracellular localization and presumably activity are regulated by chemoattractants in Dictyostelium and mouse neutrophils. However, the mechanisms for its regulation remain elusive. Here we show that RhoA and Cdc42, members of the Rho family of small GTPases, regulate the intracellular localization of PTEN in leukocytes and human transfected embryonic kidney cells. In addition, active RhoA is able to stimulate the phospholipid phosphatase activity of PTEN in human embryonic kidney cells and leukocytes, and this regulation seems to require RhoA's downstream effector, RhoA-associated kinase (Rock). Furthermore, we have identified key residues on PTEN that are required for its regulation by the small GTPase, and show that small GTPase-mediated regulation of PTEN has a significant role in the regulation of chemotaxis.

Animals↗

PubMed2004

Involvement of the C-terminal proline-rich motif of G protein-coupled receptor kinases in recognition of activated rhodopsin.

G protein-coupled receptor kinases (GRKs) are a family of serine/threonine kinases that phosphorylate many activated G protein-coupled receptors (GPCRs) and play an important role in GPCR desensitization. Our previous work has demonstrated that the C-terminal conserved region (CC) of GRK-2 participates in interaction with rhodopsin and that this interaction is necessary for GRK-2-mediated receptor phosphorylation (Gan, X. Q., Wang, J. Y., Yang, Q. H., Li, Z., Liu, F., Pei, G., and Li, L. (2000) J. Biol. Chem. 275, 8469-8474). In this report, we further investigated whether the CC of other GRKs had the same functions and defined the specific sequences in CC that are required for the functions. The CC regions of GRK-1, GRK-2, and GRK-5, representatives of the three subfamilies of GRKs, could bind rhodopsin in vitro and inhibit GRK-2-mediated phosphorylation of rhodopsin, but not a peptide GRK substrate. Through a series of mutagenesis analyses, a proline-rich motif in the CC was identified as the key element involved in the interaction between the CC region and rhodopsin. Point mutations of this motif not only disrupted the interaction of GRK-2 with rhodopsin but also abolished the ability of GRK-2 to phosphorylate rhodopsin. The findings that the CC region of GRKs interact only with the light-activated but not the non-activated rhodopsin and that the N-terminal domain of GRK-2 interacts with rhodopsin in a light-independent manner suggest that the CC region is responsible for the recognition of activated GPCRs in the canonical model.

Amino Acid Motifs↗

PubMed2004

[Construction of eukaryotic vector and expression of chimeric antibody against rh-bFGF].

AIM: To express a human-mouse chimeric antibody against rh-bFGF antigen in eukaryotic cells. METHODS: The VL and VH genes were amplified and cloned from the hybridoma 1F11 secreting anti-rh-bFGF mouse monoclonal antibody (mAb) and the CL and CH genes were amplified and cloned from the plasmid pMDHC and pMDLC. They were inserted into eukaryotic expression vectors after sequencing. The recombinant plasmids were then transformed into CHO-dhfr- cells for expression. The humanization and antigen specificity of expressed products were identified by indirect ELISA. The relative molecular mass (Mr) of expressed products was determined by SDS-PAGE. RESULTS: The cloned antibody genes were identified to be functional by sequencing. The chimeric antibody could be detected in the culture supernatant of transformed CHO cells. The humanization and specificity of the chimeric antibody to rh-bFGF were confirmed by ELISA. CONCLUSION: The mouse-human chimeric antibody against rh-bFGF has been expressed successfully in eukaryotic cells, which lays the foundation for its further clinical research.

Amino Acid Sequence↗

PubMed2004

[Purification of recombinant humanized anti-HBsAg Fab antibody by affinity chromatography].

AIM: To establish a steady purification method for producing recombinant humanized anti-HBsAg Fab from yeast fermentation supernatant. METHODS: Humanized anti-HBs scFv was used to immunize BALB/c mice to obtain anti-scFv monoclonal antibody (mAb) which was used to purify recombinant humanized anti-HBsAg Fab from yeast fermentation supernatant by affinity chromatography. RESULTS: The purity of purified recombinant anti-HBsAg Fab was above 95% and its recovery rate was about 75%-85%. CONCLUSION: An efficient affinity chromatography suitable for purification of recombinant humanized anti-HBsAg Fab in large-scale was established.

Animals↗

PubMed2004

[Production of recombinant humanized anti-HBsAg Fab antibody by fermentation].

In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.

Fermentation↗

PubMed2003

Electrochromatography with a 2.7 mm inner diameter monolithic column.

Monolithic columns of 2.7 mm I.D. have been prepared and used in electrochromatography (EC) separation. Although capillary electrochromatography (CEC) has higher separation efficiency, it displays some shortcomings, such as limited sample loadability and restricted concentration detectability etc. In this paper, we investigate the feasibility of EC separation with millimeter diameter monolithic columns. By using a designed preparation method of monolithic column packed with about 150 microm quartz sand, the effect of Joule heating can be reduced, and the processes of frit making and column packing can be avoided. The concentration detectability of the EC is improved comparing with that of CEC. Moreover, the separation efficiency of 52,000 plates/m was achieved with a 70 mm length and 2.7 mm I.D. monolithic column.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2002

[The expression of humanized Fab fragment of the anti-HBsAg antibody in methylotropic yeast Pichia pastoris].

Using of two-step integrating technology, transducted the H and L chain gene of humanized Fab fragment of anti-HB-sAg antibody into the genome of methylotropic yeast P. pastoris. Constructed a engineering yeast to produce humanized Fab fragment of the anti-HBsAg antibody. The Fab fragment was efficiently secreted into the medium at a concentration of 50-80 mg/L. The Fab fragment was purified from culturing supernatant of the recombinant yeas by affinity chromatography. The ELISA analysis showed the high affinity of the expressed humanized Fab fragment to the HBsAg.

Chromatography, Affinity↗