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 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

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

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

OBJECTIVETo investigate the antigenicity of recombinant Chlamydia trachomatis (Ct) OmcBc protein and search for the new target for early diagnosis of Chlamydia infection and Chlamydia vaccine development.

METHODSThe C fragment of OmcB encoding the amino acids from T270 to T553 was amplified from Chlamydia serovar D genomic DNA. The pGEX-6p-Ct OmcBc expression plasmid was constructed and transformed into E.coli XL-1blue. The expression of recombinant Ct OmcBc protein was induced by IPTG. Serum samples were collected from 120 patients with urogenital Chlamydia infection. The antiserum samples were collected from 7 New Zealand white rabbits and 5 Balb/C mice immunized subcutaneously and intraperitoneally with Ct serovar D inactivated EB, respectively, and from 9 Balb/C mice intranasally infected with Ct serovar D live EB. The anti-Chlamydia specific antibody were titrated by an immunofluorescence assay (IFA). The reactivity of the recombinant OmcBc protein with all the above antisera was detected by ELISA.

RESULTSThe pGEX-6p-Ct OmcBc expression plasmid was successfully constructed. DNA sequencing showed that the inserted OmcBc was about 852 bp, encoding a protein with 284 amino acids. The expression of the recombinant GST-OmcBc was induced by IPTG, producing a fusion protein with a molecular weight of about 57 kD. The titer of the specific antibodies to Chlamydia in all the antisera was high. ELISA results showed strong reactivities of the recombinant GST-OmcBc fusion protein with all the above antisera.

CONCLUSIONSOmcBc protein is an immunodominant protein of Chlamydia. The recombinant GST-OmcBc with strong antigenicity may provide a basis for further study of early diagnosis of chlamydia infection and development of Chlamydia vaccine.

Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Genes, Bacterial ; Humans ; Immune Sera ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; Rabbits

Due to the serovar diversity in Haemophilus (H.) parasuis, it is difficult to develop a universal serological method for detection of this pathogen. Here, we report a universal plate-agglutination test for detecting H. parasuis. Diagnostic antisera were prepared by mixing antisera of serovars 4, 5, 12, 13 and 14 in the optimized ratio. The results of the plate-agglutination test showed that the diagnostic antisera could agglutinate with all 15 reference strains of H. parasuis and 74/75 clinical isolates. Further, the specificity of the method was validated with 22 bacterial strains from 12 related species.
Agglutination Tests/*methods ; Animals ; Cross Reactions ; Haemophilus parasuis/isolation & purification/*physiology ; Immune Sera/*metabolism ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2.

METHODSThe coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2.

RESULTSPCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP).

CONCLUSIONThe prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Breast Neoplasms ; genetics ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Immune Sera ; biosynthesis ; Inhibitor of Differentiation Protein 2 ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.
Acrosome ; drug effects ; metabolism ; Acrosome Reaction ; physiology ; Adult ; Animals ; Calcium ; metabolism ; Fluorescent Antibody Technique, Indirect ; Humans ; Immune Sera ; pharmacology ; Male ; Mice ; Middle Aged ; Phosphoinositide Phospholipase C ; immunology ; metabolism ; Sperm Capacitation ; drug effects ; physiology ; Spermatozoa ; drug effects ; metabolism

OBJECTIVETo prepare and identify a polyclonal antibody against rat myostatin and investigate myostatin expression in the rat atrophic gastrocnemius muscle after tibial nerve crush.

METHODSThe purified fusion protein was used as antigen to immunize rabbits for the preparation of polyclonal antibody. The polyclonal antibody of the protein was measured by enzyme linked immunosorbent assay (ELISA), western-blot and immunochemistry. Myostatin protein expression levels in normal and atrophic gastrocnemius muscle were detected by western-blot and immunochemistry assays.

RESULTSThe GST-myostatin had a purity of 96% and possessed high titer and specificity. The level of myostatin in gastrocnemius muscle significantly increased one week after tibial nerve crush, reached the peak on day 14, and then returned to normal level on day 28.

CONCLUSIONWe have successfully made antiserum of rat myostatin and found that the expression level of myostatin protein in the gastrocnemius after tibial nerve crush-induced atrophy was time-dependent. This study provides an experimental basis to clarify the possible role of myostatin during skeletal muscle atrophy.

Analysis of Variance ; Animals ; Antibodies ; blood ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Gene Expression Regulation ; physiology ; Immune Sera ; Immunization ; methods ; Male ; Muscle, Skeletal ; metabolism ; Myostatin ; immunology ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; immunology ; Tibial Neuropathy ; metabolism ; pathology ; Time Factors

OBJECTIVETo express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).

METHODSThe 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.

RESULTSThe prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.

CONCLUSIONThe fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

Animals ; Antibodies ; immunology ; isolation & purification ; Antibody Specificity ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; immunology ; Membrane Proteins ; biosynthesis ; genetics ; immunology ; Nerve Tissue Proteins ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology