1.Clinical evaluation of four recombinant Treponema pallidum antigen-based rapid tests in the diagnosis of syphilis.
Lin-na WANG ; Lei YANG ; He-yi ZHENG
Chinese Medical Sciences Journal 2007;22(4):250-253
OBJECTIVETo assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigen-based rapid tests in the diagnosis of syphilis.
METHODSA total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. pallidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests. T. pallidum haemagglutination test (TPHA) was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed.
RESULTSThe sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test, VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98.9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9%; for whole blood were 74.1% and 99.5%, 87.9% and 99.4%, 73.2% and 99.7%, 64.7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA (P > 0.05).
CONCLUSIONSThe 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.
Antigens, Bacterial ; immunology ; Humans ; Quality Control ; Recombinant Proteins ; immunology ; Sensitivity and Specificity ; Syphilis ; diagnosis ; Treponema pallidum ; immunology
2.Progress in research on molecular biology and application in dominant antigens ESAT6 and CFP10 of TB vaccine.
Journal of Biomedical Engineering 2012;29(2):392-396
As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial
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chemistry
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genetics
;
immunology
;
Bacterial Proteins
;
chemistry
;
genetics
;
immunology
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Humans
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Immunodominant Epitopes
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immunology
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Molecular Biology
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Peptide Fragments
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chemistry
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genetics
;
immunology
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Tuberculosis Vaccines
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genetics
;
immunology
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Vaccines, DNA
;
immunology
3.Distribution of Salmonella paratyphi A pagC gene and immunoprotective effect of its recombinant expressed products.
Jia ZHANG ; Xinli FAN ; Yumei GE ; Jie YAN ; Aihua SUN
Journal of Zhejiang University. Medical sciences 2013;42(2):171-231
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
4.Construction of fusion gene vaccine of WT1 multi-epitope fused with stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and its expression and immunogenicity.
Wei-Wei TIAN ; Zhen-Hua QIAO ; Lin-Hua YANG ; Hong-Wei WANG ; Yan-Hong TANG ; Si-Cheng BIAN
Journal of Experimental Hematology 2011;19(2):485-490
This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.
Bacterial Proteins
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genetics
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immunology
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Epitopes
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genetics
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immunology
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Genetic Vectors
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HSP70 Heat-Shock Proteins
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genetics
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immunology
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Humans
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Immunodominant Epitopes
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Vaccines, DNA
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genetics
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immunology
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WT1 Proteins
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genetics
;
immunology
5.Prokaryotic expression and antigenicity of the CagA gene in Helicobacter pylori.
Xiong CHEN ; Canxia XU ; Fen WANG ; Shourong SHEN ; Yan JIA
Journal of Central South University(Medical Sciences) 2010;35(8):847-853
OBJECTIVE:
To synthesize the specific CagA gene segment of the gastric cancer idiotype Helicobacter pylori (H. pylori), establish the prokaryotic expression system and identify the antigenicity sequence of recombination signals.
METHODS:
We selected the CagA fragment which was related to gastric cancer in our earlier research. The CagA gene segment was optimized and synthesized. The synthesized CagA gene was cut from the pUC57-CagA plasmid and then was carried by expression vector pET32a to be transformed into the host bacterium BL21 (DE3). The positively cloned pET32a-CagA was selected by receptivity of aniline and colony PCR. The host bacterium with pET32a-CagA was induced by IPTG to express fusion protein. The expression of CagA protein was analyzed by SDS-PAGE gel electrophoresis and the antigenicity of fusion protein was examined by Western blot.
RESULTS:
CagA gene segment was designed and synthesized. The sequence of synthesis CagA gene segment was the same as the one designed before (AF289435). We successfully constructed the plasmid of prokaryotic expression of the pET32a-CagA. Homology of the target CagA proteinum was 100%, the same as AAG09884. The host bacterium BL21 (DE3) containing pET32a-CagA could express CagA fusion protein after the IPTG induction. SDS-PAGE gel electrophoresis showed that the molecular weight of fusion protein was the same as expected (45 kD). Western blot showed that the fusion protein could be combined with the antibody of the whole bacterium of anti-H. pylori.
CONCLUSION
The synthesized CagA fusion protein from the prokaryotic expression system has antigenicity. We hope to set the foundation for selecting the strain in H. pylori correlated to gastric cancer and corresponding therapy in clinical practice.
Amino Acid Sequence
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Antigens, Bacterial
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genetics
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immunology
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Bacterial Proteins
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genetics
;
immunology
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Base Sequence
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Genetic Vectors
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Helicobacter pylori
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genetics
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immunology
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Molecular Sequence Data
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Recombinant Fusion Proteins
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biosynthesis
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genetics
;
immunology
6.Expression of main protein antigen of Helicobacter pylori and serum antibody in infected patients.
Chinese Journal of Epidemiology 2004;25(9):820-820
Antibodies, Bacterial
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blood
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Antigens, Bacterial
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biosynthesis
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Bacterial Proteins
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biosynthesis
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Carrier Proteins
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biosynthesis
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Gastritis
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microbiology
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Helicobacter Infections
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immunology
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Helicobacter pylori
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immunology
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isolation & purification
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Humans
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Recombinant Proteins
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biosynthesis
7.Asymptomatic Infection by Streptococcus pyogenes in Schoolchildren and Diagnostic Usefulness of Antideoxyribonuclease B.
Journal of Korean Medical Science 2005;20(6):938-940
This study is designed to evaluate the immune status of schoolchildren with respect to Streptococcus pyogenes, and to ascertain the usefulness of antideoxyribonuclease B (ADNase B). Antistreptolysin O (ASO) and ADNase B concentrations were measured quantitatively in 266 serum samples from healthy elementary school children in Seoul. Simultaneously, throat cultures were taken in order to isolate S. pyogenes and other beta-hemolytic streptococci (BHS). The upper limits of the normal (ULN) concentration of ASO and ADNase B were 326 IU/mL, and 362 IU/mL, respectively. The correlation between ADNase B (y) and ASO (x) was y=0.4x+173 (r= 0.46). Mean ADNase B level (392 IU/mL) was significantly higher in children with S. pyogenes than in those with non-group A BHS (236 IU/mL) or no BHS (234 IU/ mL). Some schoolchildren were proven, via ASO and ADNase B tests, to be harboring asymptomatic S. pyogenes infections. The high ULN of ASO and ADNase B in schoolchildren should be carefully considered, in order to interpret the data collected from the patients. We could add the ADNase B test to our set of diagnostic tools, which would allow us to more accurately detect and diagnose streptococcal infections, as ADNase B was more specifically related to the results of throat cultures, and there was little correlation between ASO and ADNase B.
Antibodies, Bacterial/*blood
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Bacterial Proteins/immunology
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Child
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Deoxyribonucleases/*immunology
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Female
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Humans
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Korea
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Male
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Serologic Tests
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Streptococcal Infections/*diagnosis/*immunology
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Streptococcus pyogenes/enzymology/*immunology
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Streptolysins/immunology
8.Immunogenicity of multi-epitopes gene of major outer membrane protein of Chlamydia trachomatis.
Shan-li ZHU ; Zhao-hui SHI ; Wen-shu LI ; Jun CHEN ; Li-fang ZHANG
Chinese Journal of Preventive Medicine 2009;43(3):232-236
OBJECTIVETo construct a recombinant eukaryotic expression plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene, and evaluate the Ct MOMP-specific humoral and cellular immune response induced by pcDNA3.1-Ct MOMP168 in BALB/c mice.
METHODSRecombinant plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene was constructed. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1-Ct MOMP168 or pcDNA3.1 or PBS (n = 12, 100 microg/time per mouse), and the same immunization schedule was repeated for the third time at 2 week intervals. The titers of anti-Ct MOMP antibody and its antibody subtypes in sera, the cytotoxicity of Ct MOMP-specific cytotoxic T lymphocyte (CTL) in spleen, and the level of cytokine (IFN-gamma, IL-4, IL-10)-producing CD3(+) T cells in spleen were detected by ELISA, LDH release assays and intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS), respectively.
RESULTSThe recombinant plasmid pcDNA3.1-Ct MOMP168 was able to induce Ct-specific antibody response (A(490) = 0.973 +/- 0.136; serum titer was 1:1000) as compared with pcDNA3.1 (A(490) = 0.180 +/- 0.025) and PBS (A(490) = 0.110 +/- 0.015), and the major antibody subtype was IgG2a with statistical significance (F = 106.884, P < 0.05). When the ratio of effector cells and target cells reached to 50:1, the activity of cytotoxic T-lymphocyte in pcDNA3.1-Ct MOMP168 immunized mice (41.71% +/- 8.34%) was significantly higher (F = 22.315, P < 0.05) than that in pcDNA3.1 immunized mice (18.40% +/- 3.45%) and PBS immunized mice (14.50% +/- 2.42%). The levels of CD3(+) IFN-gamma(+) T cells in pcDNA3.1-Ct MOMP168 immunized mice (1.15% +/- 0.16%) were significantly higher (F = 99.638, P < 0.05) than that in pcDNA3.1 immunized mice (0.12% +/- 0.08%) and PBS immunized mice (0.09% +/- 0.03%), while the significant difference in the levels of IL-4(+) CD3(+) T cells and IL-10(+) CD3(+) T cells was not observed (F = 0.886 and 1.112, P > 0.05) between pcDNA3.1-Ct MOMP168 immunized mice (0.13% +/- 0.08% and 0.14% +/- 0.08%) and pcDNA3.1 (0.07% +/- 0.05% and 0.13% +/- 0.06%) or PBS immunized mice (0.08% +/- 0.04% and 0.07% +/- 0.04%).
CONCLUSIONIn BALB/c mice, the recombinant plasmid pcDNA3.1-Ct MOMP168 might induce not only the generation of Ct-specific antibody, but also the high level of Ct MOMP-specific CD3(+) IFN-gamma(+) T cells.
Animals ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Porins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology
9.Regulatory T-cell vaccination independent of auto-antigen.
David W PASCUAL ; Xinghong YANG ; Kathryn HOLDERNESS ; Sangmu JUN ; Massimo MADDALONI ; Irina KOCHETKOVA
Experimental & Molecular Medicine 2014;46(3):e82-
To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25+ Treg cells are stimulated, but for arthritis CD39+ Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-beta and interleukin-10.
Animals
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Antigens, Bacterial/*immunology
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Arthritis, Rheumatoid/immunology/prevention & control
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Autoantigens/*immunology
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Fimbriae Proteins/*immunology
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Humans
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Multiple Sclerosis/immunology/prevention & control
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Salmonella/*immunology
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T-Lymphocytes, Regulatory/*immunology
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*Vaccination
10.Cloning, expression and immunity of pilA gene and ompC gene from avian pathogenic Escherichia coli.
Shan YU ; Qian ZHANG ; Xiaoxi SHUI ; Zhouliang YU ; Baohua ZHAO
Chinese Journal of Biotechnology 2008;24(9):1561-1567
In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.
Animals
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Cloning, Molecular
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Escherichia coli
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genetics
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immunology
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metabolism
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Escherichia coli Proteins
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genetics
;
immunology
;
metabolism
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Escherichia coli Vaccines
;
immunology
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Fimbriae Proteins
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genetics
;
immunology
;
metabolism
;
Gene Expression Regulation, Bacterial
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Genes, Bacterial
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Mice
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Porins
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genetics
;
immunology
;
metabolism
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Recombinant Fusion Proteins
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genetics
;
immunology
;
metabolism