OBJECTIVETo construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters.

METHODSThe groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT.

RESULTSThe nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels.

CONCLUSIONThe GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

Agglutination Tests ; Animals ; Antigens, Bacterial ; immunology ; Chaperonin 60 ; genetics ; immunology ; Cricetinae ; Gene Expression ; Leptospira interrogans ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice.

METHODSAs model antigen, the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4.0 with intron A involved and pVAX1 without intron A involved, respectively. The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines. Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining.

RESULTSCompared with non-intron A pVAX1hsp65, the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAX1hsp65 caused higher expression level of Hsp65 in 293T cells, and enhanced Th1 type immune response, which was defined as higher level of anti-Hsp65 specific total IgG level (3.76 ± 0.23 vs 3.15 ± 0.22, P < 0.01) and IgG2a/IgG1 ratio (4.08 ± 0.04 vs 2.23 ± 0.12, P < 0.01) and more IFN-γ-secreting CD4(+) ((2.0 ± 0.058)% vs (1.5 ± 0.087)%, t = 4.804, P < 0.01) and CD8(+) ((0.6 ± 0.058)% vs (1.0 ± 0.115)%, t = 3.098, P < 0.05) T lymphocytes. The difference showed statistical significance.

CONCLUSIONIntron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.

Animals ; Bacterial Proteins ; genetics ; immunology ; Chaperonin 60 ; genetics ; immunology ; Female ; Genetic Vectors ; Introns ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; Vaccines, DNA ; genetics ; immunology

Behcet's disease is recognized as a systemic inflammatory disease of unknown etiology. The disease has a chronic course with periodic exacerbations and progressive deterioration. Previous reports have shown at least three major pathophysiologic changes in Behcet's disease; excessive functions of neutrophils, vasculitis with endothelial injuries, and autoimmune responses. Many reports suggested that immunological abnormalities and neutrophil hyperfunction may be involved in the etiology and the pathophysiology of this disease. HLA-B51 molecules by themselves may be responsible, in part, for neutrophil hyperfunction in Behcet's disease. T cells in this disease proliferated vigorously in response to a specific peptide of human heat shock protein (hsp) 60 in an antigen-specific fashion. T cells reactive with self-peptides produced Th1-like proinflammatory and/or inflammatory cytokines. This leads to tissue injury, possibly via delayed-type hypersensitivity reaction, macrophage activation, and activation and/or recruitment of neutrophils. These data shed new light on the autoimmune nature of Behcet's disease; molecular mimicry mechanisms may induce and/or exacerbate Behcet's disease by bacterial antigens that have activated T cells which are reactive with self-peptide(s) of hsp. This would lead to positive selection of autoreactive T cells in this disease.
Behcet's Syndrome/pathology ; Behcet's Syndrome/immunology* ; Behcet's Syndrome/etiology ; Chaperonin 60/immunology ; Eye/pathology ; Human ; Neutrophils/physiology ; Skin/pathology ; T-Lymphocytes/physiology

OBJECTIVETo determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.

METHODSOmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.

RESULTSThe bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01).

CONCLUSIONExpression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cell Line ; Chaperonin 60 ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; pathogenicity ; Lipoproteins ; genetics ; metabolism ; Macrophages ; microbiology

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.
BCG Vaccine ; biosynthesis ; immunology ; Bacterial Proteins ; biosynthesis ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; immunology ; Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Mycobacterium tuberculosis ; genetics ; immunology ; Plasmids ; genetics ; Sequence Analysis, DNA ; Vaccines, Synthetic ; biosynthesis ; immunology

Heat shock proteins (HSP) have been identified as an important factor of a very complex and highly conserved cellular defense mechanism to preserve cell survival under adverse environmental conditions. HSP 60 are immunodominant antigens of microbe such as Chlamydia trachomatis and have a potentiality to become a target antigen due to antigenic similarity between chlamydial and human HSP. This study was conducted to investigate the effects of Vero cell coculture to anti-HSP 60 on the early mouse embryo development in vitro. The 2-cell mouse embryos (ICR) were cultured and mouse embryo development was observed every 24 hr for 3 days. 45% and 22.1% of the embryos cultured in Ham's F-10 plus anti HSP 60 with Vero cells developed to the 4- to 8- cell stage (day 1) and morular stage (day 2) as compared with 29.2% and 2.7% of those cultured without Vero cells respectively. But at day 3, the beneficial effect of Vero cells was not noted. These findings suggest that Vero cells have some roles to overcome the detrimental effect of anti-HSP 60 to some degree. These results suggest that Vero cells coculture will promote reproductive outcome in patient previously sensitized to microbial (e.g. Chlamydia trachomatis) HSP 60.
Vero Cells ; Pregnancy ; Mice, Inbred ICR ; Mice ; Male ; Infertility, Female/etiology/immunology/therapy ; Immunodominant Epitopes ; Female ; Embryonic Development/*immunology ; Coculture Techniques ; Chlamydia trachomatis/immunology/pathogenicity ; Chaperonin 60/*immunology ; Cercopithecus aethiops ; Antigens, Bacterial ; Antibodies, Monoclonal/*administration & dosage ; Animals

OBJECTIVETo evaluate the therapeutic effect of MHSP65-TCL on melanoma and its effect on the activity of the immunocytes.

METHODSMHSP65-TCL was prepared by mixing MHSP65 with TCL derived from B16 melanoma cell lysate by repeated freezing and thawing. The MHSP65-TCL vaccine was administered in mice bearing B16 melanoma, and the changes in melanoma growth was observed. To investigate the influence of TCL in MHSP65-TCL on the activity of the immunocytes, we co-cultured TCL and mouse spleen cells in vitro, and analyzed CD69 expression on the cells, cell apoptosis, and levels of IL-10 and IFN-γ in the cell culture supernatant.

RESULTSThe MHSP65-TCL vaccine showed an anti-melanoma effect in the tumor-bearing mice. In the in vitro experiment, TCL in MHSP65-TCL strongly stimulated the activation of mouse spleen cells while causing apoptosis in some of the immunocytes and promoting cellular IL-10 secretion, but not IFN-γ.

CONCLUSIONSMHSP65-TCL derived from B16 melanoma cells has an anti-melanoma effect mediated by the activation of immunocytes. TCL in MHSP65-TCL also has immunosuppressive effect on immunocytes possibly due to the presence of suppressive components in TCL, and identifying and eliminating these components may potentially improve the anti-tumor actovoty of MSHP65-TCL vaccine.

Animals ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Apoptosis ; Bacterial Proteins ; administration & dosage ; immunology ; Cancer Vaccines ; Cell Extracts ; administration & dosage ; immunology ; Cell Line, Tumor ; Chaperonin 60 ; administration & dosage ; immunology ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Lectins, C-Type ; metabolism ; Melanoma, Experimental ; immunology ; pathology ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Spleen ; cytology ; immunology ; metabolism ; Tumor Burden ; immunology

To improve the efficacy of peptide P277 in preventing autoimmune diabetes, heat shock protein 65 kD (HSP65) of Mycobacterium tuberculosis var. bovis was fused with linear polypeptide epitope of P277 and expressed as soluble protein in Escherichia coli. The fusion protein HSP65-P277 was purified by anion exchange column chromatography and then used to immunize prediabetic NOD mice with three ip inoculations in absence of adjuvants. Serum samples from the immunized mice were collected monthly and the concentration of blood glucose was measured. The study showed that administration of HSP65-P277 to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself or HSP65. Fused to heat shock protein 65 of Mycobacterium tuberculosis could improve the efficacy of diabetes prevention of P277 in nonobese diabetic mice. The results suggest the fusion protein of HSP65-P277 would be useful for treating insulin-dependent diabetes mellitus.
Animals ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; immunology ; Diabetes Mellitus, Type 1 ; prevention & control ; Escherichia coli ; genetics ; metabolism ; Female ; Heat-Shock Proteins ; genetics ; immunology ; Immunization ; Mice ; Mice, Inbred NOD ; Mycobacterium bovis ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology

OBJECTIVETo investigate the influence of endothelial dysfunction induced by inoculated dendritic cells (DCs) loaded heat shock protein 60 (HSP60) in apolipoprotein (Apo) E-null mice, and the effect of Puerarin on it.

METHODSHSP60 DC (DChsp) acquired after prepared bone marrow-derived DCs of ApoE-null mice and treated with HSP60. In vitro, the function of DCs and the effect of Puerarin were detected. While in vivo, ApoE-null mice fed with high-cholesterol forage were divided into two groups and intravenous inoculated with DCh-sp or normal saline via vein twice respectively. The mice in the two groups were subdivided into the Puerarin group and non-treated group, and they were injected intraperitoneally with Puerarin and normal saline at the beginning of inoculation and the following 3 weeks, respectively. In addition, C57BL/6 mice without inoculation were taken as the normal control group. Two weeks after the last time inoculated, the response of T lymphocytes to HSP60 and endothelial-dependent diastolic function of aortic ring were detected.

RESULTSHSP60 could promote DCs expressed CD86 and stimulate T lymphocytes proliferation in vitro, while Puerarin had significantly inhibitory effect. After inoculated, DChsp activated inflammatory response in vivo and aggravated endothelium-dependent dilation in mice. Puerarin could significantly inhibit inflammatory reaction caused by DChsp and improve endothelium dilation.

CONCLUSIONHsp60 could activate DCs in vitro and in vivo, Puerarin could significantly inhibit specific immunity induced by HSP60 and improve vascular endothelium-dependent dilation.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Apolipoproteins E ; genetics ; B7-2 Antigen ; immunology ; Cell Proliferation ; drug effects ; Chaperonin 60 ; metabolism ; Dendritic Cells ; drug effects ; enzymology ; immunology ; Endothelium, Vascular ; drug effects ; physiology ; Immunity ; drug effects ; Inflammation ; chemically induced ; Isoflavones ; pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Protective Agents ; pharmacology ; T-Lymphocytes ; drug effects ; Vasodilator Agents ; pharmacology

OBJECTIVETo fuse Legionella virulence gene (lvgA) with heat shock protein 60 gene (Hsp60) by PCR and detect the fusion gene expression in E.coli.

METHODSThe fragments of lvgA and Hsp60 genes having matching sequences at their ends to be fused were amplified from the genomic DNA of Legionella pneumophila by PCR, and the PCR products were mixed, denatured, reannealed, so that the strands with matching sequences at their 3' ends overlapped to serve as primers for each other. Extension of this overlap by DNA polymerase produced recombinant products. After amplification with outer primers, sufficient product of the fusion gene was harvested, which was inserted into the prokaryotic expression vector pGEX-4T-1 to construct the prokaryotic expression recombinant plasmid. After identification with restriction enzyme analysis, polymerase chain reaction and nucleotide sequence analysis, the E.coli BL21 containing the recombinant plasmid pGlvgA/Hsp60 was induced with IPTG and the expression of lvgA/Hsp60 was detected by SDS-PAGE and Western blot analysis.

RESULTSThe lvgA/Hsp60 fusion gene of 2,292 bp was amplified and the recombinant plasmid pGlvgA/Hsp60 was constructed successfully. A 117-kD GST-lvgA-Hsp60 fusion protein was detected in the E.coli containing the recombinant plasmid.

CONCLUSIONThe recombinant plasmid for Legionella pneumophila lvgA/Hsp60 fusion gene is constructed successfully and this fusion protein can be expressed in prokaryotic cells efficiently, which make possible the immunological characterization of this fusion gene.

Alternative Splicing ; Chaperonin 60 ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Gene Fusion ; Genetic Vectors ; Humans ; Legionnaires' Disease ; immunology ; prevention & control ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Vaccines, DNA ; biosynthesis ; immunology ; Virulence Factors ; biosynthesis ; genetics