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Biomedical subjects

Bo Niu

Publications and source records attributed to Bo Niu.

5 recordsLinked to original sources
PubMed2006

[Fermentation and purification of Echistatin fusion protein expressed in Escherichia coli].

Lots of studies of Echistatin (Ecs) have proved its wide use in many aspects. However, the low yield of Ecs has impeded the relative researches of the protein. To establish the high-level expression system of Ecs, the fermentation and purification process of Ecs fusion protein expressed in E. coli were optimized. The Ecs gene was introduced into vector pTXB1 and placed under the control of highly efficient T7 promoter system. The cloned Ecs gene was expressed in E. coli BL21 (DE3) as soluble form. The Ecs production and biomass accumulation were optimized by examining medium composition, point of induction and induction time in fed-batch fermentation. Biomass accumulation was greatly affected by medium gradient, reaching 50.3g/L in 2 x YT medium. Ecs production was found to increase to 35% of total protein with 75g/L biomass accumulation after induced for 4h. Purification of Ecs from supernatant of sonication was done using one-step chromatographic procedure with chitin affinity chromatography and DTF cleavage, resulting in yields of 28mg/L and > 90% purity. The bioactivity of purified Ecs was determined and the result showed that purified Ecs could inhibit the aggregation of platelet in vitro with similar bioactivity to wild Ecs. This optimized method is readily scaled up for the expression and purification of Ecs in sufficient quantities for further structural and biological studies and applications.

Cloning, Molecular↗
PubMed2006

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen.

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable (ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg, the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay (ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy (VH) and variable light (VL) and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 x 10(6) and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Antibodies, Monoclonal↗
PubMed2005

Protein transduction domain of membrane penetrating peptide can efficiently deliver DNA and protein into mouse liver for gene therapy.

BACKGROUND: The development of a harmless and efficient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of this study was to test a nonviral gene transferring vector's function of delivering DNA into liver cells to provide an important clue for gene transfer in liver gene therapy. METHODS: The complex of DNA and DNA delivering protein was injected into mice through their tail veins. Then the mice were killed and their liver tissue was sectioned. The gene transferring results were detected using a confocal laser scanning microscope. RESULTS: Fluorescence analysis indicated that both DNA-membrane penetrating peptide (MPP) complex and DNA- hepatocyte specific receptor binding domain (HSRBD)-MPP complex could go into liver cells. The fluorescence value of liver cells in the DNA- HSRBD-MPP group was higher than that in the DNA-MPP group. CONCLUSIONS: MPP can successfully deliver DNA and protein into cells, and MPP with a HSRBD can specifically deliver DNA into liver cells. These have laid a foundation for further study on the nonviral liver cell gene delivering system.

Animals↗
PubMed2004

Structure analysis of the protein transduction domain of human Period1 and its mutant analogs.

Human Period1 (hPer1) has been proved to be able to translocate into cells in a protein transduction manner. The segment of amino acids 830-845 of hPer1 is its protein transduction domain (PTD). In order to explore the membrane penetrating mechanism of hPer1-PTD and the physico-chemical properties necessary in the process, Ala scanning mutation method was used to investigate the variation in the peptide internalization. To further investigate the related physico-chemical requirements, the three dimensional structures of hPer1-PTD and its mutant analogs were simulated by Rosetta method. The electrostatic potentials and energies of these structures were calculated using the Delphi algorithm to solve Poisson-Boltzman equation. The hydrophobicity was assessed by the percentage of the nonpolar area in SAS (solvent accessible surface (SAS)). It has been proved that the Arg836 was the key residue for peptide internalization. When this Arg mutated into Ala, the peptide could not cross the membrane. The large enough area with positive charge was the decisive factor for hPer1-PTD. The alpha-helical structure seemed to play an assistant role so as to enable the positive charge connected in spatial arrangement.

Amino Acid Sequence↗