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Objective The truncated fibronectin-binding proteins A (Fba protein) were cloned and expressed, then animals were immunized with Fba protein and subsequently challenged with group A streptococcus (GAS) to further investigate protective antibodies induced by each domain and determine the most immunogenic domain. Methods Fba proteins, which were divided into four overlaps based on the structural domains, were truncated and expressed. The fba truncated genes were amplified by polymerase chain reaction (PCR) with SSI-9 of GAS as template, and cloned into prokaryotic expression plasmid pGEX-2T and expressed in E. coli BL-21. The products were confirmed by Western blot and purified by affinity chromatography column. Female BALB/c mice were immunized with the four truncated proteins respectively, with phosphate buffered solution (PBS) as control. The serum IgG of mice was detected by enzyme-linked immunosorbent assay (ELISA). After the third immunization, mice were challenged with fatal dose of GAS (M+ Fba+ ) to evaluate the protective rates in each group. The data were compared by analysis of variance and Fisher's exact test.Results Prokaryotic expression plasmids of pGEX-2T/FbaAl, pGEX-2T/FbaA2, pGEX-2T/FbaA3 and pGEX-2T/FbaA4 were successfully constructed and the four truncated proteins were expressed and purified successfully. Serum levels of IgG in each experimental group gradually increased with immunization with Fba protein more times. After the third immunization, the IgG titer against FbaA2 1290.2, P<0. 01). After GAS challenge, four out of eight mice were protected in FbaA2 protein group, while two out of eight mice in FbaAl protein, FbaA3 protein and FbaA4 protein groups,respectively (P<0. 05). Conclusions Four truncated Fba proteins are constructed and expressed successfully. Truncated FbaA2 protein could be able to induce strongest protective immune response.
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Objective:To identify monoclonal antibody McAb2 recognizing epitope of Fba of GAS.Methods:The overlapped peptides were synthesized and their abilities to bind McAb2 were detected by dot-ELISA.The predominance amino acids specific for McAb2 were screened using phage 7 peptide library.Results:The result by dot-ELISA analysis demonstrated that the synthetized peptide,amino-acid residues 100-112~(th),could bind McAb2 with high affinity.The predominance amino acids specific for McAb2 were ITPDL,which was located in 100-110~(th)aa of Fba by panning with phage 7 peptide library.Conclusion:The domain and the predominance amino acids of Fba recognized by McAb2 is determined.The results would contribute to the research of the role of Fba on the pathogenic mechanism of GAS,the identification of function of McAb2,and the development of epitope-peptide vaccine.
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Objective To express the novel fibronectin-binding protein Fba of group A streptococcus(GAS)and analyze its immunogenicity,so that to evaluate the immune responses to GAS infection.Methods fba gene was amplified by polymerase chain reaction(PCR)and confirmed by sequencing.Then it was cloned into pGEX4T-2 vector and Fba protein was expressed in E.coli BL21.The protein expression was identified by enzyme-linked immunosorbent assay(ELISA)and Westernblot.The sera from mice infected with GAS and anti-streptolysin-O positive patients were detected using microtiter plates coated with purified Fba protein as antigen.Afterward Balb/C mice were immunized with this purified protein and the sera were collected after the third immunization for the detection of IgG titer.Results It was confirmed by ELISA and Western blot that the recombinant Fba protein had a specific affinity with anti-Fba sera of rabbit.The anti-serum IgG titer of mice imrnunized with Fba protein was up to 1:4800.Conclusions GAS infection or Fba protein immunization are able to induce high serum titer of anti-Fba which could react specifically with the recombinant Fba protein.It indicates that Fba protein has good immunogenicity and antigenicity.So Fba protein could be a GAS candidate vaccine and an important tool to detect anti-GAS titer in GAS infected patients.
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<p><b>BACKGROUND</b>To study the correlation among the number of tumor-infiltrating dendritic cells (TIDC) in cancer tissues and biological behavior and prognosis in lung cancer patients.</p><p><b>METHODS</b>S-100 protein expression level was determined in 39 patients with lung cancer by immunohistochemistry technique. The number of S-100 + TIDC and DNA ploidy were measured by means of flow cytometry.</p><p><b>RESULTS</b>The rate of positive S-100 protein expression was 100% in 39 patients, S-100 + cells showed typical morphology of dendritic cells. The percentage of S-100 +TIDC in patients with heteroploid (21.81%±8.18%) was significantly higher than those with diploid (16.03%±4.75%) (P=0.006). There was no statistical difference between lymph node metastasis group (20.43%±7.74%) and no lymph node metastasis group ( 19.41% ±7.76%), between tumor size greater than 3cm group ( 20.90% ±8.65%) and less than 3cm group ( 19.70% ±7.61%), between non-small cell lung cancer group (19.48%±7.98%) and small cell lung cancer group (21.74%±6.17%). No correlation was found between survival time ( 1 year , 1-3 years, greater than 3 years, respectively) and percentage of S-100 +TIDC (21.96%±8.05%, 19.47%±6.18%, 19.14%±8.76%, respectively).</p><p><b>CONCLUSIONS</b>The number of TIDC should not be chosen as an independent prognostic criterion in human lung cancer.</p>
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Objective:To observe cellular,humoral and mucosal immune responses induced by DNA-or protein-based of FimH of UPEC type 1.Methods:After mice were immunized respectively with recombinant plasmid pcDNA3.1/fimH or pcDNA3.1/fimC,and the combinant FimH and FimC protein,the anti-FimH protein IgG of sera and SIgA in bladders were detected by ELISA.The lymphocyte phenotypes of CD3,CD4 and CD8 were analyzed by FCM.Results:The titers of IgG in sera and SIgA in the bladders were all low in the group immunized by recombinant FimH plamid,but the percentage of CD4+T cells in spleen was high,which revealed that recombinant FimH plamid was able to trigger better cellular immune response.The titers of IgG were very high in the group immunized by FimH protein,which suggested that the FimH protein was able to trigger better humoral immune response,but SIgA in the bladders was not detectable.Conclusion:The DNA for FimH can induce humoral,mucosal and cellular immune response.FimH protein can only induce humoral immune response.FimC protein is able to enhance the immunogenicity of FimH protein.