ABSTRACT
A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.
Subject(s)
Inclusion Bodies/chemistry , Inclusion Bodies/genetics , Protein Folding , Receptors, Cytokine/genetics , Recombinant Fusion Proteins/isolation & purification , Animals , Antibody Specificity , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/immunology , Chromatography, Affinity/methods , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Humans , Immunoglobulin Variable Region/genetics , Mice , Mice, Inbred BALB C , Receptors, Cytokine/chemistry , Receptors, Cytokine/isolation & purification , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , T-Lymphocytes/immunology , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). METHODS: Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). RESULTS: This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. CONCLUSION: This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.
Subject(s)
RNA, Viral/analysis , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/isolation & purification , Child, Preschool , Humans , Infant , Nasopharynx/virology , Polymerase Chain Reaction/methodsABSTRACT
We combined the specificity of tumor-specific antibody with the chemokine function of interferon-gamma inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V(H) region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.
Subject(s)
Antibody Specificity , Immunoglobulin Fragments/metabolism , Receptors, Cytokine/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Base Sequence , Cell Line , DNA Primers , Genetic Vectors , Immunoglobulin Fragments/genetics , Mice , Protein Binding , Receptors, Cytokine/genetics , Receptors, Cytokine/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , T-LymphocytesABSTRACT
A functional single-chain Fv antibody fragment (scFv) specific for acidic isoferritin (AIF) was produced at high level in Escherichia coli. The variable regions of heavy chain (V(H)) and light chain (V(L)) from the hybridoma 4c9 were connected with a flexible linker using an assembly polymerase chain reaction. The construct of V(H)-linker-V(L) was inserted into a phagemid pCANTAB 5 E followed by selection with the Recombinant Phage Antibody System (RPAS). Anti-AIF scFv gene from the recombinant phagemid pCAN4c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active anti-AIF scFv was recovered efficiently from inclusion bodies with a refolding yield of approximate 75% confirmed by spectrophotometer. The activity of refolded scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed anti-AIF scFv retains the specific binding activity to AIF with an affinity constant of 7.29 x 10(-8) mol l(-1). The overall yield of anti-AIF scFv with bioactivity in E. coli flask culture was more than 60 mg l(-1).
