ABSTRACT
Background Researches demonstrated that CpG ODN,a immunostimulatory sequences,has preventing and treating effect on allergic conjunctivitis caused by protein allergen.However,its effect on allergic conjunctivitis caused by fungal allergen is unclear. Objective This study aimed to investigate into whether the Th1-Th2 switching immunostimulatory CpG ODN could reverse the response in the murine allergic conjunctivitis model caused by aspergillus fumigatus. Methods A mixture of spores and hyphae of aspergillus fumigatus strain was used to induce allergic conjunctivitis in male BALB/C mice aged 6-8 weeks.This experiment was designed into preventive or therapeutical treatment program.Under both settings,allergic conjunctivitis of the animals were treated with CpG ODN,nonstimulatory GpC ODN or PBS.After the last challenge with the allergen,the clinical symptoms of the animals were scored based on the criteria of Magone.The animals were sacrificed and the histopathological examination of conjunctiva was performed.Expression of TLR4 mRNA in conjunctiva was analyzed by real-time PCR assay.The responsiveness and populations of lymphocytes in spleen and draining lymph nodes were analyzed by flow cytometry.The use complied with the Standard of Association for Research in Vision and Ophthalmology. Results In the prevention mode.CpG ODN decreased subconjunctival infiltration compared with GpC ODN and PBS groups with the average neutrophil count index(21.25 ±11.59/section,30.75 ±11.44 section and 69.00±9.90/section,respectively).Expression of TLR4 mRNA was up-regulated significantly by CpG ODN.The clinical scores for CpG ODN group were insignificantly lower than those in GpC ODN group and PBS group(P＞0.05).In the therapeutic mode,compared with GpC ODN and PBS groups,the allergic symptom score in CpG ODN group manifested significantly lower(t=4.000.t=2.750,P＜0.01)and showed fewer cellular infiltration(t=4.870,t=3.829,P＜0.01)and higher expression of TLR4 mRNA(P＜0.01).In cultured splenic and draining lymph node cells,increased percentages of CD4+ CD25+ and CD4+ CD25+ CD69+ in CpG ODN group were observed compared with control groups(|P＜0.05). Conclusion CpG ODN can relieve aspergillus fumigatus-induced allergic conjunctivitis via either subconjunctival injection or topical application by upregulating expression of TLR4 and activating Treg lymphocytes.
ABSTRACT
The full length cry2Ab gene was cloned by PCR-RFLP method from Bt strain B-Pr-88, which was isolated in China with high toxicity to the Lepidopteran insect pests. Nucleic acid sequence analysis showed that this gene was 1902 base pairs encoding 633 amino acids. This cry gene was named cry2Ab4 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The full open reading frame sequence of the cry2Ab4 gene was amplified with a pair of PCR primers L2ab5/L2ab3 designed according to its DNA sequence,and inserted into the BamH I /EcoR I sites of E. coli expression vector pET21b to obtain the recombinant plasmid pET-2Ab4. The result of SDS-PAGE proved that Cry2Ab4 could be expressed as a 60 kD protein in E. coli BL21 (DE3)strain induced by IPTG. Bioassay of the expressed product of the cry2Ab4 gene showed that Cry2Ab4 was highly toxic to the larvae of Helicoverpa armigera and Leguminivora glycinivorella, moderately active to the larvae of Plutella xylostella and Chilo suppressalis, but not insecticidal to the larvae of Spodotera exigua and Ostrinia furnacalis. Our result indicated that cry2Ab4 gene could be used as a novel gene for generation of transgenic plants and engineered microorganism.
Subject(s)
Bacillus thuringiensis , Genetics , Bacterial Proteins , Genetics , Cloning, Molecular , Endotoxins , Genetics , Escherichia coli , Genetics , Metabolism , Genes, Bacterial , Hemolysin Proteins , Genetics , Pest Control, Biological , Recombinant Proteins , Genetics , Sequence Analysis, DNAABSTRACT
<p><b>OBJECTIVE</b>To observe the immunogenicity of combined hepatitis A and B vaccine (HAB).</p><p><b>METHODS</b>The combined HAB vaccine was prepared and different concentrations of HAB were administered on mice in week 0, 4 and 24, and then we tested the antibodies to both hepatitis A virus and B virus. After the first injection, we tested the hepatitis A antigen-induced and hepatitis B surface antigen-induced stimulation indices in spleen monocyte as well as changes of CD4+ and CD8+ cell numbers.</p><p><b>RESULTS</b>The serum antibody positive rates were 100% in all three groups, and the antibody induced by HAB vaccine were earlier than by monovalent vaccine. The hepatitis A antibody and hepatitis B surface antibody titers after the combined vaccine inoculation were not significantly higher than those after the monovalent vaccine inoculation. On the other hand, after the first injection of the combined vaccine, the hepatitis A antigen-induced and hepatitis B surface antigen-induced stimulation indices in spleen monocyte were detected. The numbers of CD4+ and CD8+ cells increased.</p><p><b>CONCLUSIONS</b>HAB vaccine has reliable immunogenicity.</p>