IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.
Algal Proteins ; biosynthesis ; immunology ; Animals ; Antibodies ; chemistry ; Blotting, Western ; Chlamydomonas reinhardtii ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Fluorescent Antibody Technique ; Intracellular Signaling Peptides and Proteins ; biosynthesis ; immunology ; Plasmids ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Cell Line ; Chimera ; Cloning, Molecular ; Humans ; Lentivirus ; genetics ; metabolism ; Mice ; Receptor, ErbB-2 ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection

OBJECTIVETo prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus).

METHODSThe recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria.

RESULTSThe fusion protein was successfully expressed, and the titer of the antibody reached 1:32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP.

CONCLUSIONThe antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.

Animals ; Antibodies, Bacterial ; biosynthesis ; genetics ; Antibody Specificity ; Bacterial Proteins ; immunology ; Helicobacter hepaticus ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction

Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL.
Animals ; CHO Cells ; Cricetulus ; Epitopes ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Vaccines ; biosynthesis ; genetics ; Hepatitis B virus ; Protein Precursors ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. FXN is a mitochondrial protein which plays an important role in the regulation of intracellular iron trafficking, biogenesis of iron-sulfur cluster and heme, and removal of reactive oxygen species. Our previous work showed that tissue-specific expression of FXN in cerebellum and heart generates two novel isoforms. In order to find the isoforms in mouse tissues, we tried to obtain a polyclonal antibody against mouse Fxn with high specificity and sensitivity. Thus, the recombinant plasmid pET24(+)-mFxn was constructed to express his-tagged Fxn in BL21 (DE3) cells. The expressed protein is a mature form with 130 amino acids (aa, 14.38 kDa) without the N-terminal signal peptide (77 aa), purified on Ni-NTA column and further dialyzed with Centrifugal Filtration Device. The polyclonal antibody against Fxn was produced by immunizing rabbits with highly purified protein. The collected antiserums were preliminarily purified by precipitation with (NH4)2SO4. Western blotting analysis and cell immunofluorescence showed that the obtained antibody was able to detect both purified and endogenous Fxn. It also worked well in immunoprecipitation with mouse tissues. This is the first time, to our knowledge, to report that mouse Fxn was used as immunogen to generate antibody with high specificity and sensitivity. This work provides a powerful tool for our further research on mouse Fxn isoforms.
Animals ; Antibodies ; immunology ; metabolism ; Antibody Specificity ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Engineering ; methods ; Immunization ; Iron-Binding Proteins ; genetics ; immunology ; Mice ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.
Animals ; Female ; Gene Products, tat ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo study the effects of immunization with the fusion protein CAC (a product of prokaryotic expression of recombinant HBcAg and β-amyloid peptide fusion gene) against the toxicity induced by intrahippocampal injection of aggregated β-amyloid peptide (Aβ) in rats.

METHODSSD rats were immunized intraperitoneally with the fusion protein CAC, and the titer of anti-Aβ antibody was evaluated by ELISA. When the titers of the anti-Aβ antibody reached 1:3 000, aggregated Aβ was injected into the CA1 region of the rat hippocampus. Two weeks after Aβ injection, the rats underwent morris water maze test before sacrificed to prepare the brain slices with Congo red and haematoxylin staining.

RESULTSThe titer of anti-Aβ antibody reached 1:3 000 after 5 immunizations with the fusion protein. After Aβ injection, the saline-immunized rats showed a reduced cognitive behavior in the Morris water maze test compared to the CAC-immunized rats. In the saline-immunized rats, the neurons around the site of Aβ injection exhibited obvious cell damages with Aβ deposits and glial infiltration, whereas in CAC-immunized rats, Aβ deposits were significantly reduced or even absent.

CONCLUSIONImmunization with the fusion protein CAC can inhibit the toxicity induced by intrahippocampal aggregated Aβ injection.

Amyloid beta-Peptides ; administration & dosage ; biosynthesis ; genetics ; immunology ; Animals ; Antibodies ; blood ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hippocampus ; metabolism ; Immunization ; Injections ; Male ; Peptide Fragments ; administration & dosage ; immunology ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells.

METHODSpORF8 gene was amplified and cloned into pGEX-6p vector, and the pORF8 fusion protein was expressed in E.coli XL1 Blue. After purification with glutathione-conjugated agarose beads, the pORF8 fusion protein was used to immunize BALB/c mice to generate polyclonal antibodies against pORF8 protein. The antibodies obtained were used to localize the plasmid protein pORF8 in Chlamydia-infected cells with immunofluorescence assay (IFA).

RESULTSThe pORF8 gene 744 bp in length was successfully cloned and the GST fusion protein with a relative molecular mass of 54 000 was obtained. The cellular distribution pattern of the plasmid protein pORF8 was similar to that of the major outer membrane protein (MOMP), a known C. trachomatis inclusion body protein, but not to that of chlamydial protease-like activity factor (CPAF, a secreted protein).

CONCLUSIONThe plasmid protein pORF8 is localized on the bacterial organism as an inclusion body protein in C. trachomatis-infected cells. The cellular location of pORF8 protein can potentially provide important insights into the pathogenesis of C. trachomatis.

Animals ; Antibodies ; immunology ; Bacterial Proteins ; biosynthesis ; genetics ; Chlamydia Infections ; metabolism ; Chlamydia trachomatis ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Secretory IgA (SIgA) antibodies in external secretions play an important role in mucosal immune response. Polymeric SIgA was advantageous over monomeric IgA (mIgA) and IgG in several aspects. To express secretory IgA antibody against H5N1 virus, we constructed the secretory component and immunoglobulin J expressing plasmids and co-transfected the plasmids into the Chinese hamster ovary cells (CHO) stably expressing immunoglobulin A. Then we used Zeocin to select the positive clone cells, monoclonal cells stably secreting SIgA was screened through fold dilution method at last. The SIgA antibody secreted from the CHO cells was confirmed by Western blotting, which demonstrated that we had got the complete SIgA molecular. The successful expression of this polymeric anti-H5N1 SIgA in CHO cells will contribute to the production of recombinant SIgA as a preventive agent for infectious disease control.
Animals ; Antibodies, Viral ; biosynthesis ; genetics ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Immunoglobulin A ; immunology ; Immunoglobulin A, Secretory ; biosynthesis ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-beta-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.
Animals ; Animals, Newborn ; Bordetella bronchiseptica ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mice ; Neutralization Tests ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transglutaminases ; biosynthesis ; genetics ; Virulence Factors, Bordetella ; biosynthesis ; genetics