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1.
Sheng Wu Gong Cheng Xue Bao ; 23(1): 40-5, 2007 Jan.
Article in Chinese | MEDLINE | ID: mdl-17366886

ABSTRACT

This study was aimed to establish ELISA for recombinant bovine IFN-gamma (BovIFN-gamma) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-gamma (BovIFN-gamma) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-gamma. The pETBovlFN-gamma was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-gamma as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-gamma were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG1 . Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-gamma, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-gamma specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-gamma, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-gamma paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.


Subject(s)
Antibodies, Monoclonal/immunology , Interferon-gamma/immunology , Recombinant Proteins/immunology , Animals , Animals, Newborn , Antibodies, Monoclonal/biosynthesis , Antibody Specificity/immunology , Blotting, Western , Cattle , Cells, Cultured , DNA, Complementary/genetics , Enzyme-Linked Immunosorbent Assay/methods , Female , Hybridomas , Interferon-gamma/genetics , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Plasmids/genetics , Rabbits , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction
2.
Sheng Wu Gong Cheng Xue Bao ; 21(2): 315-9, 2005 Mar.
Article in Chinese | MEDLINE | ID: mdl-16013497

ABSTRACT

In order to differently diagnose avian influenza virus (AIV) subtypes, the HA gene of AIV H9 subtype was cloned, expressed and utilized in an enzyme-linked immunoad sorbent assay (ELISA). HA gene (1683bp) of H9N2 AIV was amplified by RT-PCR from a strain of field isolated H9N2 AIV, and its identity was confirmed by sequencing. The HA gene was subcloned into prokaryotic expression vector pGEX-KG with its secretion signal sequence removed. The expressed HA-GST fusion protein in E. coli BL21 was characterized by SDS-PAGE and western blotting analysis as a 90kD protein with immunogenicity. The fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation. The HA-GST fusion protein was used to establish an indirect ELISA for the detection of antibodies to H9 subtypes of AIV. The assay has 91.57% specificity to H9 AIV, 92.31% sensitivity and excellent reduplication. It could be used to differently detect antibodies to H9 AIV.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H9N2 Subtype/genetics , Influenza, Human/diagnosis , Recombinant Proteins , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/biosynthesis , Humans , Influenza, Human/virology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
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