OBJECTIVETo obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression, purification, and immunogenicity.

METHODSThe Mycobacterium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction, and then cloned into the vector pTA2, followed by the subclone into the expression vector pET30a (+). The resulting plasmids, named pET30a (+): rv1837c and pET30a (+): rv3803c, encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a (+): rv1837c and pET30a (+): rv3803c were introduced into E. coli BL21 (DE3) by transformation respectively, and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western blot with hexa-histidine tag antibody and serum from tuberculotic patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin. Rabbits were immunized with purified recombinant Rv1837c and Rv3803c proteins. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbit serum, which had been immunized by Western blot.

RESULTSAfter transformation of the E. coli and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside, recombinant target proteins Rv1837c (relative molecular mass: 92000) and Rv3803c (relative molecular mass: 38 000) were expressed in pET30a (+): rv1837c and pET30a (+): rv3803c system. The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins of E. coli. The purity of the purified protein reached 90%. The immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong, as identified by Western blot.

CONCLUSIONThe prokaryotic expression recombinant plasmids pET30a (+): rv1837c and pET30a (+): rv3803c was successfully constructed and the recombinant proteins Rv1837c and Rv3803c were obtained, which laid a basis for the optimized diagnosis of active tuberculosis.

Antibodies ; metabolism ; Bacterial Proteins ; genetics ; immunology ; metabolism ; Blotting, Western ; Escherichia coli ; metabolism ; Genetic Vectors ; Mycobacterium tuberculosis ; genetics ; immunology ; metabolism ; Plasmids ; metabolism ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVETo determine the location on outer envelope and natural antibody response and types of genus-specific lipoprotein antigen LipL41s in patients with Leptospira interrogans.

METHODSMicroscope agglutination test (MAT) was used to examine leptospirosis patients' serum samples from Sichuan area, China. Ni-NTA affinity chromatography was performed to extract the target recombinant rLipL41/1 and rLipL41/2 products that expressed under inducement of IPTG. Western blot assay was performed to detect the immunoreactivity between the sera from the patients infected with different serogroups of L. interrogans and rLipL41s. Immune aurosol electron microscopy was selected to locate the position of LipL41s on leptospiral envelope. ELISA based on rLipL41s was established to confirm the level and types of specific antibody.

RESULTSL. interrogans serogroup icterohaemorrhagiae remained to be the most dominant leptospiral serogroup in Sichuan area. All the sera from patients infected with different serogroups of L. interrogans could efficiently recognize the LipL41s which were the protein molecular that located on the external surface of leptospiral envelope. In the 156 serum samples from MAT positive leptospirosis patients, the positive rates for rLipL41/1 or rLipL41/2 specific IgM appeared to be 84.6%-87.8% and 78.2%-83.3%, respectively, while for rLipL41/1 or rLipL41/2 specific IgG they were 69.2%-81.4% and 75.0%-80.1%, respectively.

CONCLUSIONLipL41s were the leptospiral superficial protein antigen of L. interrogans. Both the LipL41/1 and LipL41/2 could induce serum antibodies IgM and IgG with extensive antigenic-cross reaction during natural infection of L. interrogans in general populations. Hence, rLipL41/1 or rLipL41/2 could be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.

Antibodies, Bacterial ; blood ; immunology ; Antigens, Bacterial ; metabolism ; Bacterial Outer Membrane Proteins ; metabolism ; Cell Membrane ; metabolism ; Cross Reactions ; Humans ; Immunoglobulin G ; blood ; immunology ; Immunoglobulin M ; blood ; immunology ; Leptospira interrogans ; metabolism ; ultrastructure ; Leptospirosis ; immunology ; Species Specificity

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

A meta-analysis was performed on the immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines after 2 (2 and 4 months) and 3 doses (2, 4, and 6 months) in Korean infants. A database search of MEDLINE, KoreaMed, and Korean Medical Database was done. The primary outcome measure was the proportion of infants with anti-polyribosylribitol phosphate (PRP) concentrations > or =1.0 microgram/mL. Eight studies including eleven trials were retrieved. One trial reported on the diphtheria toxoid conjugate vaccine (PRP-D) and 2 trials each on the mutant diphtheria toxin (PRP-CRM) and Neisseria meningitidis outer-membrane protein (PRP-OMP) conjugate vaccine. Heterogeneity in study designs between trials on PRP-CRM was noted and one trial reported on a monovalent and another on a combination PRP-OMP vaccine. Thus, a meta-analysis was conducted only on the tetanus toxoid conjugate vaccine (PRP-T). After a primary series of 2 doses and 3 doses, 80.6% (95% confidence interval [CI]; 76.0-85.1%) and 95.7% (95% CI; 94.0-98.0%) of infants achieved an antibody level > or =1.0 microgram/mL, respectively. The immunogenic response to the PRP-T vaccine was acceptable after a primary series of 3 doses and also 2 doses. A reduced number of doses as a primary series could be carefully considered in Korean infants.
Antibodies/analysis ; Bacterial Capsules/*immunology/metabolism ; Haemophilus Vaccines/*immunology/metabolism ; Humans ; Infant ; Republic of Korea ; Tetanus Toxoid/chemistry/metabolism ; Vaccines, Conjugate/immunology/metabolism

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

OBJECTIVETo explore etiology, clinical manifestation and immunological changes of infectious pneumonia of neonates in Chengdu area.

METHODSSerum specimens were collected from 111 infants with infectious pneumonia. Eight viral and mycoplasmal specific serum IgM antibodies were detected by enzyme linked immunosorbent assay (ELISA); C reactive protein (CRP), total IgG and its subclasses, IgA and IgM were determined by rate scattered nephelometry; T lymphocyte subpopulations were detected by biotin-streptavidin-peroxidase method, and clinical and other laboratory data were analyzed.

RESULTS(1) Etiological agents: specific serum IgM antibodies were positive in 40 of 111 cases (36.0%) with pneumonias. All the 30 control infants were negative for the specific serum IgM antibodies. Among 111 infants with infectious pneumonia, 20.7% had single viral or mycoplasmal infection, 40.5% had bacterial infection, 15.3% had viral and mycoplasmal infection with bacterial infection; 23.4% had infection with unknown agents. (2) The most common clinical manifestations were tachypnea and cyanosis. The next were cough, milk choking, rales, retractions of the supraclavicular, intercostal and subcostal areas. Roentgenographic examination commonly revealed vague opacities, increased density and patchy infiltration. (3) Immune status: (1) CD(3), CD(4) cell counts of infants with pneumonias were lower than those of the controls while their serum IgA, IgM concentrations were higher than those of the control. (2) The CD(3) and CD(4) cell counts of the group with bacterial infection were lower than those of the control group. (3) The serum IgA concentration of the group with viral and mycoplasmal infection was higher than those of the control group and the group with unknown infection. (4) The serum IgM concentration of the group with bacterial infection was higher than those of the control group. (5) There were no significant differences in CD(8) cell counts, CD(4)/CD(8), concentration of serum IgG and IgG(1 - 4) between pneumonia group and the control group, and among various infectious groups and the control.

CONCLUSIONPathogens of neonatal infectious pneumonia in Chengdu area included single viral or mycoplasmic infection or bacterial infection, viral and mycoplasmal infection with bacterial infection, and unknown infection. Immunological changes of newborn infants suffered from infectious pneumonia included declined CD(3) and CD(4) cell counts, particularly in bacterial infection.

Antibodies, Bacterial ; blood ; Antibodies, Viral ; blood ; Bacterial Infections ; complications ; C-Reactive Protein ; analysis ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin M ; blood ; Infant, Newborn ; Male ; Pneumonia ; diagnosis ; etiology ; immunology ; T-Lymphocyte Subsets ; immunology ; metabolism ; Virus Diseases ; complications

OBJECTIVESTo study the effect of monoclonal antibody (McAb) against helicobacter pylori (Hp) ureB, 1F11 on platelet aggregation and activation, and its mechanism.

METHODSThe relativity between human platelet glycoproteins (GPs) and Hp ureB was identified by Western blot and FCM. Platelet aggregation was measured by turbidimetry, and P-selectin and TXB2 assay by ELISA.

RESULTS1F11 could bind to platelet GPIIIa, and ADP-induced platelet aggregation was inhibited by 1F11 in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 induced by ADP. The FCM results show that the positive rates of platelet binding to FITC-SZ21 was decreased from 99.5% to 77.4% after addition of 1F11.

CONCLUSIONMcAb against Hp ureB 1F11 inhibits platelet aggregation through binding to platelet GPIIIa but does not block platelet activation. There might be crossed-epitopes on Hp ureB and platelet GPIIIa, and Hp infection might be involved in ITP immunopathology.

Antibodies, Bacterial ; pharmacology ; Antibodies, Monoclonal ; pharmacology ; Bacterial Proteins ; immunology ; metabolism ; Helicobacter pylori ; immunology ; Humans ; Integrin beta3 ; immunology ; P-Selectin ; immunology ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Urease ; immunology ; Urokinase-Type Plasminogen Activator ; immunology

OBJECTIVETo evaluate the effects of Chlamydia pneumoniae infection on oxidative stress and the development of atherosclerosis in C57BL/6J mice.

METHODSForty-eight C57BL/6J mice were divided into 4 groups including infection of CP and cholesterol diet, cholesterol diet, infection of CP and control. Atherosclerotic lesions were measured in the aortic root at 40 weeks after the primary infection. Production of superoxide was measured by lucigenin-enhanced chemiluminescence response and evaluated in situ with laser scanning confocal microscope.

RESULTSInfected mice fed with an atherogenic diet developed significantly larger lesion areas compared with the single atherogenic diet mice (135 249 +/- 43 748 microm2 vs. 96 378 +/- 30 945 microm2, P < 0.05). Superoxide generation was higher in aortic arches of the infected mice or atherogenic diet mice compared with the control mice (1974.25 +/- 650.49, 701.00 +/- 105.16, 455.62 +/- 77.54 counts.mg(-1).min(-1) vs. 142.25 +/- 31.82 counts.mg(-1).min(-1), respectively, P < 0.001).

CONCLUSIONChlamydia pneumoniae infection accelerates atherosclerotic lesion development in diet-induced hypercholesterolemic mice. Generation of reactive oxygen species may contribute to atherosclerotic development by Chlamydia pneumoniae infection.

Animals ; Antibodies, Bacterial ; blood ; Atherosclerosis ; etiology ; Chlamydia Infections ; complications ; metabolism ; Chlamydophila pneumoniae ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Superoxides ; metabolism

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

LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.
Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enterotoxins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; Female ; Gene Fusion ; genetics ; Mice ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism