To construct a recombinant Bacillus subtilis strain expressing SpaA and CbpB of Erysipelothrix rhusiopathiae for oral administration, we constructed the recombinant plasmid pDG1730-CBJA by fusion PCR and seamless cloning. The plasmid was introduced into B. subtilis KC strain by natural transformation, and the recombinant strain KC-spaA-cbpB was screened out on the plate containing spectinomycin (spe^r) and confirmed by PCR and starch degradation test. The SpaA and CbpB expressed by KC-spaA-cbpB were detected by Western blotting and indirect immunofluorescence assay, and the genetic stability of the recombinant strain in mice was determined. The plasmid pMAD-∆spe^r with knockout of spe^r was constructed and transformed into KC-spaA-cbpB. The spe^r-deleted mutant strain KC-spaA-cbpB: : ∆spe^r was screened and identified, and its immunogenicity in a mouse model was evaluated by oral immunization. The results showed that the recombinant strain KC-spaA-cbpB was stable in mice, expressing SpaA on the cell surface and CbpB on the spore surface. KC-spaA-cbpB: : ∆spe^r expressed SpaA and CbpB. The mice vaccinated with the spores of KC-spaA-cbpB: : ∆spe^r had higher levels of SpaA and CbpB-specific IgG in the serum that those vaccinated with the wild-type spores 42 days after vaccination by gavage (P < 0.01). The protective rate of mice immunized with the recombinant spores was 67.5%. The results indicated that a recombinant B. subtilis strain expressing SpaA and CbpB of E. rhusiopathiae was successfully constructed, and the recombinant strain laid a foundation for the development of oral live vector vaccines for swine erysipelas.
Animals ; Bacillus subtilis/immunology* ; Mice ; Erysipelothrix/immunology* ; Bacterial Proteins/immunology* ; Bacterial Vaccines/genetics* ; Erysipelothrix Infections/prevention & control* ; Immunization ; Mice, Inbred BALB C ; Plasmids/genetics* ; Immunogenicity, Vaccine ; Administration, Oral ; Antigens, Bacterial

The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.
Animals ; Antigens, Bacterial/*immunology ; Brucella abortus/*enzymology/immunology ; Brucellosis/diagnosis/*veterinary ; Cattle ; Cattle Diseases/*diagnosis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli/genetics ; Malate Dehydrogenase/*genetics/*immunology/isolation & purification ; Mice ; Recombinant Proteins/genetics/*immunology

Lipooligosacharide (LOS) of Neisseria gonorrhoeae (gonococci, GC) is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide (alpha-OS) moiety of LOS (lgtF mutant) significantly impairs invasion of GC into epithelial cell lines. GC opacity (Opa) proteins, such as OpaI, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen (CEA) family, which includes CEACAM3 (CD66d), a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of OpaI-expressing lgtF mutant on phagocytosis by HeLa-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgtF mutant even expressing OpaI completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in HeLa cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgtF mutant, which might result from the conformational change, cannot be functional.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; metabolism ; Carbohydrate Sequence ; Carcinoembryonic Antigen ; genetics ; immunology ; Gene Expression Regulation ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Lipopolysaccharides ; chemistry ; immunology ; Luminescent Measurements ; Mutation ; Neisseria gonorrhoeae ; genetics ; metabolism ; pathogenicity ; Neutrophils ; immunology ; microbiology ; Phagocytosis

OBJECTIVETo investigate superantigen gene profiles of group A Streptococcus (GAS) isolated in Beijing pediatric patients in 2014, and to explore the the relationship between superantigen gene profiles with emm types, and GAS infections with diseases.

METHODSA total of 259 GAS strains were isolated from pediatric patients clinically who diagnosed with scarlet fever and pharyngitis from 36 hospitals in Beijing from May to July, 2014.The Superantigens genes of strains were performed by Real-time PCR (speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ, ssa). PCR amplification of GAS strain M protein N gene segments were carried ort; products after sequencing comparison were analyzed to determine the GAS types of emm. The differences in distributions of superantigen genes and emm types of GAS isolates were compared between subgroups.

RESULTSAmong the 259 GAS strains, the detection rates of 13 superantigens were as the following: speA 48.6% (126), speB 99.2% (257), speC 99.2% (257), speF 98.8% (256), speG 98.5% (255), speH 43.6% (113), speI 46.3% (120), speJ 49.0% (127), smeZ 99.2% (257) and ssa 98.5% (255), respectively, however, speK, speL, and speM were not found. Eleven superantigen gene profiles in all were observed (A-K). The percentage of emm1 strains harbored spe A and speJ were 94.2% (113/120), 95.0% (114/120), respectively, which were significantly higher than those of emm12 strains (5.6% (7/124), 5.6% (7/124), respectively; χ(2) = 191.20, 194.80, P < 0.001). The percentage of emm12 strains harbored speH and speI were 83.9% (104/124), 88.7% (110/124), respectively, which were significantly higher than those of emm1 strains (3.3% (4/120), 4.2% (5/120), respectively; χ(2) = 160.30, 174.90, P < 0.001).The superantigen genotypes of GAS strains and emm types, which were isolated from scarlet fever and pharyngitis cases, were not significant different (P > 0.05).

CONCLUSIONThe GSA strains isolated in Beijing pediatric patients in 2014, the relevance ratio of speB, speC, speF, smeZ, speG, ssa were higher than others, while speK, speL, and speM were no detected in any GAS strains. The superantigen genes appeared to be associated with the emm type. Furthermore, emm type distribution and superantigen genes were not different between scalet fever and pharyngitis.

Antigens, Bacterial ; genetics ; Beijing ; Child ; Genotype ; Humans ; Real-Time Polymerase Chain Reaction ; Streptococcal Infections ; microbiology ; Streptococcus pyogenes ; genetics ; immunology ; Superantigens ; genetics

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Escherichia coli ; HIV Antigens ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV Protease ; genetics ; immunology ; Herpes Simplex Virus Vaccines ; immunology ; Herpesvirus 1, Human ; physiology ; Plasmids ; Transfection ; Vero Cells ; Virus Replication ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlandia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlandia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlandia and Sao Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.
Animals ; Antigens, Bacterial/blood/*diagnostic use ; Bacterial Outer Membrane Proteins/genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; Base Sequence ; Brazil ; Dog Diseases/diagnosis/*microbiology ; Dogs ; Ehrlichia canis/*genetics/*immunology/isolation & purification ; Ehrlichiosis/diagnosis/microbiology/*veterinary ; Fluorescent Antibody Technique, Indirect/veterinary ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; RNA, Ribosomal, 16S/genetics/metabolism ; Sequence Alignment/veterinary

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.
*Antigenic Variation ; Antigens/*genetics/immunology/metabolism ; Bacterial Proteins/genetics/metabolism ; Bordetella pertussis/*genetics/isolation & purification/*metabolism ; Genes, Bacterial ; Genotype ; Humans ; Pertussis Toxin/genetics/metabolism ; Promoter Regions, Genetic ; Republic of Korea ; Sequence Analysis, DNA ; Whooping Cough/immunology/*microbiology/pathology

Recent studies show that the vector of recombinant Bacillus Calmette-Guérin (rBCG) has a series of advantages. With exogenous gene and vaccine in one inoculation, it can obtain strong and persistent immune response at one time so that BCG is considered as a kind of ideal vector for live recombinant vaccine. This review outlines the application of rBCG vaccine and its vector in infectious diseases caused by bacteria, viruses, other microorganisms and parasites.
AIDS Vaccines ; genetics ; immunology ; Antigens, Bacterial ; genetics ; immunology ; BCG Vaccine ; genetics ; immunology ; metabolism ; Communicable Disease Control ; methods ; Genetic Vectors ; genetics ; HIV Infections ; prevention & control ; HIV-1 ; Mycobacterium tuberculosis ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Tuberculosis ; prevention & control ; Vaccines, Synthetic ; immunology

OBJECTIVETo determine the distribution and sequence conservation of pagC gene in Salmonella paratyphi A isolates, and the immunogenicity and immunoprotection of its recombinant expression products (rPagC).

METHODSThe distribution of pagC gene in Salmonella paratyphi A isolates and its sequence conservation were examined by PCR and sequencing. A prokaryotic expression system of pagC gene was constructed and the expressed rPagC was extracted by Ni-NTA affinity chromatography. SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rPagC. The antigenicity and immunoreactivity of rPagC were detected by immunodiffusion test, ELISA and Western Blot assay. The immunoprotective effect of rPagC against infection of Salmonella paratyphi A in mice was determined, while the agglutinative effect of sera from rPagC-immunized mice was measured by micro-Widal's test.

RESULTSAll the Salmonella paratyphi A isolates tested had the pagC gene, the similarity of nucleotide and amino acid sequences was 99.1 %-100 % and 98.4 %-100 %, respectively. The constructed prokaryotic expression system expressed rPagC with high efficiency. The rPagC immunized rabbit produced a high level antibody and it also combined with antiserum against whole cell of S. paratyphi A to generate a positive Western hybridization signal. ELISA results indicated that 97.1 % (66/68) paratyphoid patients infected with Salmonella paratyphi A were positive for rPagC antibody in their serum specimens. When mice were immunized with 100 μg or 200 μg rPagC, the immunoprotective rates were 73.3 % (11/15) or 86.7 % (13/15), respectively. The sera from rPagC-immunized mice offered 1:10-1:40 agglutination titers with the H antigens of Salmonella paratyphi A and Salmonella typhi.

CONCLUSIONPagC gene has an extensive distribution in Salmonella paratyphi A isolates. rPagC can be used as the candidate antigen in genetic engineering vaccine due to its fine immunogenicity and powerful immunoprotective effect.

Agglutination Tests ; Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Proteins ; genetics ; immunology ; Bacterial Vaccines ; Membrane Proteins ; genetics ; immunology ; Mice ; Rabbits ; Recombinant Proteins ; genetics ; immunology ; Salmonella paratyphi A ; genetics ; immunology ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid

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