The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

To explore the impact of the history of infection by the influenza A virus subtype H1N1 on secondary infection by the influenza A virus subtype H9N2, pigs non-infected and pre-infected with H1N1 were inoculated with H9N2 in parallel to compare nasal shedding and seroconversion patterns. Unlike pigs without a background of H1N1 infection, nasal shedding was not detected in pigs pre-infected with H1N1. Both groups generated antibodies against H9N2. However, levels of H1N1 antibodies in pigs pre-infected with H1N1 increased quickly and dramatically after challenge with H9N2. Cross-reaction was not observed between H1N1 antibodies and H9N2 viruses. These findings suggest that circulation of the H1N1 virus might be a barrier to the introduction and transmission of the avian H9N2 virus, thereby delaying its adaptation in pigs.
Animals ; Antibodies, Viral ; immunology ; Cross Reactions ; Immune Sera ; immunology ; Influenza A Virus, H1N1 Subtype ; immunology ; physiology ; Influenza A Virus, H9N2 Subtype ; immunology ; Orthomyxoviridae Infections ; blood ; immunology ; Species Specificity ; Swine ; immunology ; virology

Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.
Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Escherichia coli ; Flow Cytometry ; Immune Sera ; Immunoglobulin G ; blood ; Opsonin Proteins ; immunology ; Phagocytosis ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

OBJECTIVETo prepare polyclonal antibodies against sodium pump alpha 2 subunit M1-M2 extramembrane fragment (NKAα2 EM1) for studying the pathogenesis of hypertension.

METHODSAccording to the GenBank data, the amino acid sequence of NKAα2 EM1 was obtained and the target peptide (LAAMEDEPSNDN) was synthesized using a peptide synthesizer with Fmoc method and purified with high-performance liquid chromatography. The synthesized peptide was then coupled to KLH for immunizing New Zealand white rabbits for 4 times to obtain the antiserum. The IgG antibodies against the synthetic peptide, after affinity purification with Protein A, were used for detecting NKAα2 EM1 expression in rat aortic vascular smooth muscle cells by enzyme-linked immunosorbent assay and immunocytochemistry (ICC).

RESULTSThe synthesized peptide fragment , which consisted of 13 amino acid residues including one derivatized cysteine residue in the N-terminal (LAAMEDEPSNDN-C), had a theoretical relative molecular mass of 1408.48 D with a measured relative molecular mass of 1407.90 D and a purity exceeding 85.5%. The titer of the antiserum was more than 1:512 000, and the purified IgG antibody concentration was 0.965 mg/ml after purification with Protein A. At a 1:1000 dilution (final concentration of 1 µg/ml), the titer of the purified IgG antibody was more than 1:256 000. The purified IgG antibody could be used at 1:100 to 1:200 dilutions for for immunocytological examination of formalin-fixed cells.

CONCLUSIONThe anti-NKAα2 EM1 polyclonal antibodies obtained can be used in ELISA and immunocytochemistry for detecting the sodium pump alpha 2 subunit in formalin-fixed tissue or cells to facilitate investigation of the relationship between sodium pump and hypertension.

Amino Acid Sequence ; Animals ; Antibodies ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Hypertension ; Immune Sera ; Immunoglobulin G ; Immunohistochemistry ; Peptide Fragments ; Rabbits ; Rats ; Sodium-Potassium-Exchanging ATPase ; immunology

OBJECTIVETo prepare rabbit anti-mouse zona pellucida 2 (mZP2) polyclonal antibodies and test their immunoactivity.

METHODSRecombinant proteins of mZP2 expressed in Rosetta transformant was separated by SDS-PAGE, and the gel strips containing the recombinant mZP2 were cut out and emulsified to immunize New Zealand white rabbits. The antibody response of the antiserum was detected by ELISA, and the specificity of the antiserum was verified by immunohistochemical assay. The effect of the antiserum on the binding of oocytes with acrosomal reacted sperm was tested by sperm-egg binding assay.

RESULTSELISA results showed that the immunized rabbit produced anti-mZP2 antiserum. The antiserum reacted specifically with the zona pellucida of mouse ovarian sections. Sperm-egg binding assay showed that treatment of the oocytes with the anti-mZP2 antiserum caused decreased binding of zona pellucida with the acrosomal reacted sperm by 43.7%.

CONCLUSIONWe obtained rabbit anti-mouse ZP2 polyclonal antibodies that can inhibit the binding of oocytes with acrosomal reacted sperm.

Animals ; Antibodies ; immunology ; Antibody Formation ; Egg Proteins ; immunology ; Female ; Immune Sera ; Male ; Membrane Glycoproteins ; immunology ; Mice ; Oocytes ; Rabbits ; Receptors, Cell Surface ; immunology ; Recombinant Proteins ; immunology ; Sperm-Ovum Interactions ; Spermatozoa ; Zona Pellucida Glycoproteins

To compare immunological characteristics of Extracellular fibrinogen-binding protein (Efb) and Clumping factor A (CfA) of Staphylococcus aureus, we constructed two prokaryotic expression vector pET28a-Efb and pET28a-ClfA. After prokaryotical expression and purification, Efb and ClfA were used to immunize experimental animal. After the second immunization the antisera were collected and the antibody titers, the bacteria binding activity and adhesion inhibition activity of these antisera were detected by enzyme linked immunosorbent assay, adhesion inhibition assay and challenge. Both Efb and ClfA had Fibrinogen binding activity whereas the former had better Fibronectin binding activity. The bacteria binding capability of antisera of rabbits immunized with ClfA was better than that with Efb (P < 0.01). Both antisera of Efb and ClfA could inhibit adherence activity of Staphylococcus aureus to Fibrinogen and Fibronectin adherence compare to the control group (P < 0.01), and Efb had better adhesion inhibition activity than ClfA. The antibody titer of immunized group could reach 1:40 500. After the second immunization, both Efb and ClfA had good protective efficacy. This result constitutes a good foundation for Staphylococcus aureus subunit vaccine development.
Animals ; Antibodies, Bacterial ; blood ; Bacterial Adhesion ; Bacterial Proteins ; immunology ; Cattle ; microbiology ; Coagulase ; immunology ; Enzyme-Linked Immunosorbent Assay ; Fibrinogen ; metabolism ; Genetic Vectors ; Immune Sera ; immunology ; Immunization ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.
Animals ; Antibodies, Bacterial/blood/*immunology/therapeutic use ; Bacterial Proteins/immunology/therapeutic use ; Bacterial Toxins/immunology/therapeutic use ; Clostridium Infections/microbiology/prevention & control/*veterinary ; Clostridium difficile/*immunology ; Enterotoxins/immunology/therapeutic use ; Female ; Horse Diseases/microbiology/*prevention & control ; Horses ; Immune Sera/*immunology ; Immunization, Passive/*veterinary ; Mice ; Mice, Inbred C57BL ; Spores, Bacterial/immunology

To prepare recombinant human retinol binding protein 4 (RBP4) by using the baculovirus expression system and to detect its immunogenicity, the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual(pFBd), and the corresponding recombinant transfer plasmid was transformed into E. coli strain DH10bac, after transposition recombinant shuttle bacmid was screened out. The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated. After amplification of recombinant baculovirus, the recombinant baculovirus seeds were obtained. To express human RBP4, logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression. Finally, to prepare a batch of RBP4 protein, logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification. Finally for preparation of polyclonal antibody and evaluation of immunogenicity, the recombinant hRBP4 from insect cells and from E. coli were immunized rabbits. Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly, and subsequently recombinant RBP4-bacmid was generated successfully. SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa. The recombinant RBP4 protein could be secreted into the medium efficiently, and the expression level was calculated amount of 100 mg/L. Finally the rabbit antiserum was harvested after recombinant RBP4 immunization, therein the titer of antiserum against baculovirus recombinant RBP4 is 1:100 000 whereas the titer of antiserum against E. coli recombinant RBP4 is only 1:10 000. Overall, human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system, and high affinity antiserum was obtained. A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit.
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Humans ; Immune Sera ; Insecta ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Retinol-Binding Proteins, Plasma ; biosynthesis ; immunology ; Sf9 Cells ; metabolism ; Transfection

OBJECTIVESynthesis and identification of complete antigen of rutin, the traditional Chinese medicine active ingredient, and develop rapid detection of rutin using enzyme-linked immunoassay method (ELISA). Immunogenicity of the complete antigen was also studied.

METHODPrepare the complete antigen by sodium periodate solution and identified by UV scanning and SDS-PAGE test. Male New Zealand white rabbits were immunized by the antigen to obtain the antiserum.

RESULTThe results of UV analysis showed that the coupling ratio of complete antigen is 13: 1. SDS-PAGE display of the artificial antigen was delayed compared with bovine serum protein. The titer of rutin antibody is 1:4 000. The sensitivity of IC50 was 5.37 mg x L(-1), the lowest detection limit was 1 mg x L(-1), the average recovery was 102%, the intra and interspecific RSD were less than 10%, cross-reactivity rate of antibodies and other analogs were less than 1%.

CONCLUSIONRutin complete antigen was synthesized successfully, and the rapid detection of rutin by ELISA method was successfully established.

Animals ; Antibody Specificity ; immunology ; Antigens ; immunology ; Cattle ; Cross Reactions ; immunology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Immune Sera ; immunology ; Immunization ; Male ; Periodic Acid ; chemistry ; Rabbits ; Rutin ; chemical synthesis ; immunology ; Serum Albumin, Bovine ; immunology ; Solutions ; chemistry

OBJECTIVETo construct and express human CD96 gene outer membrane domain (hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.

METHODShCD96om was amplified by RT-PCR from the peripheral blood of patients with acute myeloid leukemia and inserted into prokaryotic expression vector pET32a(+) to construct the recombinant plasmid pET32-CD96. The expression of hCD96om was induced by IPTG in BL21(DE3) cells, and the expression product was identified by Western blotting. The anti-hCD96 polyclonal antibody was prepared by immunization of rabbits with the fusion protein. The specificity of anti-hCD96 antibody was determined by Western blotting.

RESULTShCD96om protein was expressed in E.coli BL21(DE3) cells in the form of inclusion body, with a relative molecular mass around 37 kD. Western blotting showed a specific reaction of the prepared antiserum with the 70 kD protein extracted from human leukemia cell line HL-60 cells and with the 37 kD hCD96om fusion protein.

CONCLUSIONThe CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells, and its rabbit polyclonal antibody has been obtained.

Animals ; Antibodies ; immunology ; metabolism ; Antigens, CD ; biosynthesis ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; biosynthesis ; Immunization ; Leukemia, Myeloid, Acute ; immunology ; Molecular Sequence Data ; Neoplastic Stem Cells ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology