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In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.
Subject(s)
Amino Acid Sequence , Bacterial Proteins , Chemistry , Chromatography , Dexamethasone , Chemistry , Molecular Weight , Pseudomonas alcaligenesABSTRACT
AIM:To evaluate the effects of treatment with HP 1188-immunoglobulin yolk ( HP1188-IgY) on Helicobacter pylori ( H.pylori)-infected gastritis in BALB/c mice.METHODS:BALB/c mice were used to establish an animal model of H.pylori-infected gastritis, and the mice were divided into 8 groups (10 mice per group).Oral antibiotics were used in group 1, 1 mg HP1188-IgY in group 2, 1 mg HP1188-IgY plus 30%sucralfate in group 3, 5 mg HP1188-IgY in group 4, 5 mg HP1188-IgY plus 30%sucralfate in group 5, PBS in group 6, and 30% sucralfate in group 7 with the treatment once per day for 10 d;and 2.5 mg HP1188-IgY was injected hypodermically twice with a 48-h interval in group 8.Another 10 mice were used as normal control in group 9.The planting of bacteria in the stomach was assayed by bacteri-al culture, rapid urease test, PCR and pathological sectioning .RESULTS:Intragastric administration with 1 mg HP1188-IgY plus 30%sucralfate per day effectively cured the injury of gastric mucosa caused by H.pylori infection, and the effect has no significant difference compared with antibiotics (P>0.05).CONCLUSION:We establish a BALB/c mouse mod-el infected with H.pylori successfully.Sucralfate (30%) is an ideal protectant for HP1188-IgY, which might decrease H. pylori infection in the stomach of BALB/c mice by oral inoculation .
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To enable students to better grasp the basic skills of pathogenic biology and im-munology experimental teaching , and make full use of the characteristics of experimental teaching to train students' scientific quality and innovative consciousness , the reform of pathogenic biology and immunology experiment teaching was explored. Microbiology, Parasitology and Immunology experiment were integrated into an experimental course , and corresponding laboratory was set up to take an in-dependent experimental teaching. Through renewing experiment teaching idea, some measures were taken such as modularization and personalization of the teaching content, the establishment of a com-plete management system , writing a new experimental course to match the experiment , improving teaching methods and developing students' innovative experiments to improve their enthusiasm and in-terest for experimental class learning, thus enhancing their innovation ability.
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<p><b>OBJECTIVE</b>To explore the relationship between Helicobacter pylori (H. pylori) infection and lower esophageal diseases in light of the changes of the bacterial flora in the lower esophagus.</p><p><b>METHODS</b>Thirty BALB/C mice were randomized into negative control group and H. pylori infection group, and in the latter group, the mice were subjected to intragastric administration of solution containing H. pylori. After 4 weeks of administration, all the mice were sacrificed, and the V6 areas in 16S rDNA were amplified from the bacterial DNA extracted from the lower esophagus using polymerase chain reaction-denaturing gradient gel electrophoresis. The bacterial floras were analyzed on DGGE atlas with Quantity-One 1-D analysis software, and the differential bands between the two groups were amplified using a 16S rDNA v6 area primer followed by DNA sequencing and BLAST analysis.</p><p><b>RESULTS</b>DGGE finger-prints showed a significantly greater number of DNA bands in the infection group than in the negative control group (P<0.01). The diversity index and richness index were also significantly higher in the infection group (0.01<P<0.05). Bacterium cluster class analysis well separated the dendrogram in the infection group. Principal component analysis showed that different groups of bacteria gathered in different locations, and BLAST analysis revealed the presence of special bacteria in the infection group.</p><p><b>CONCLUSION</b>In normal mice, Lactobacillus and the Bacteroides are the predominant bacterial flora colonizing in the lower esophagus, and Staphylococcus, Acinetobacter and Bacteridium become the predominant bacteria after H. pylori infection.</p>
Subject(s)
Animals , Mice , Bacteria , Classification , DNA, Bacterial , Esophagus , Microbiology , Helicobacter Infections , Microbiology , Helicobacter pylori , Mice, Inbred BALB C , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
The CrdS protein responding to the acidic adaptation was prokaryotic-expressed in our Laboratory to explore the regulatory mechanism in the acidic adaptation of Helicobacter pylori (H. pylori). The whole genomic DNA of H. pylori strain 26695 was abstracted and set as the template firstly. And then the hp1364 gene coding CrdS protein was amplified via the PCR technique. Then the clonal recombinant plasmid pUCm-T-hp1364 and the prokaryotic expression plasmid pQE30-hp1364 were built and identified by the methods of PCR, cutting with two enzymes and sequencing. After that, the plasmid pQE30-hp1364 was transferred into the E. coli XL1 blue and induced with IPTG. Using western blot and SDS-PAGE, it can be analyzed that the expressed recombinant protein existed mainly in the form of the inclusion bodies and its relative molecular mass was about 46 kDa. The successfully attained recombinant protein CrdS will provide the material to explore the regulatory mechanism in the acidic adaptation of H. pylori and the new way to resist the infection of H. pylori.
Subject(s)
Bacterial Proteins , Genetics , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Metabolism , Helicobacter pylori , Genetics , Plasmids , Polymerase Chain Reaction , Recombinant Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To explore the induction of L-forms of Mycobacterium abscess using isoniazid.</p><p><b>METHODS</b>Mycobacterium abscess were cultured in aqueous culture media in the presence or absence of 128 µg/ml isoniazid. The culture media containing isoniazid were filtered with 0.45 µm membrane, and the filtrate was subcultured in nutrient agar media for reversion. The isoniazid-free and isoniazid-containing media and the reversion bacteria were observed for cell wall integrity by cell wall staining and transmission electron microscopy, and the microstructures of the cell surfaces were observed by scanning electron microscopy. The isoniazid-containing culture was subcultured in L-form agar media, and the isoniazid-free culture and the reversed bacteria in nutrient agar media to observe the colony morphology. The reversed and non-induced bacteria were identified for 16S rDNA.</p><p><b>RESULTS</b>The bacteria induced with 128 µg/ml isoniazid showed cell wall defect, presenting with a spherical cell morphology and typical fried egg-like colonies in L-form agar media, while in isoniazid-free cultures, the cells showed intact cell walls with rod-like shapes and round colony morphologies on nutrient agar. The reversed bacteria, showing also intact cell walls with rod-like shapes and round colonies on nutrient agar, had identical 16S rDNA with the non-induced bacteria.</p><p><b>CONCLUSION</b>Isoniazid can induce the L-forms of mycobacterium abscess for use in studies of multidrug resistance and treatment of the bacteria.</p>
Subject(s)
Cell Wall , Culture Media , Isoniazid , Pharmacology , L Forms , Mycobacterium tuberculosis , Cell BiologyABSTRACT
To fully incarnate"specialization"peculiarities of microbiology teaching courses by means of optimizing microbiology teaching contents and curriculum systems,and making up a new syllabus suitable to different specialties will lay a very solid foundation for cultivating medical talents with specialty traits.
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In the experiments, various media were used to isolate Campylobacter Pyloridis. Onto every medium serum, blood, vitamin, glucose, charcoal and starch were added separately. The results were compared with direct Gram's stain method and urease test. Better growth was obtained on the brain-heart blood agar; almost no. growth occured on the serum agar, normal blood agar and C. jejuni agar. The positive rate was increased after charcoal and starch were added. The positive rate was significantly increased after vitamin and glucose were added. The colonies were increased significantly. It was similar with the results of the direct Gram's stain method
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Objective:To explore the immune response in mice fed orally with the conjugated antigen of HpaA-CtxB(HCTB).Methods:A recombinant strain which could express bivalent antigen of HpaA and CtxB subunit was constructed. HpaA and CtxB gene was amplified by PCR. The DNA products of HpaA and CtxB were inserted into a prokaryotic expression vector pQE-30 respectively, and then translated into E.coli strain DH5? to express HCTB fusion protein. Its immunogenicity was analyzed by Western blot. After purification, to fed mice by oral immunization. The change of antigen-specific ASC antibody(IgG and IgA) in the mucosa of the animals were detected by ELISPOT and ELISA assay. Results:HCTB fusion gene was sequenced as 1 161 bp, the fusion protein encoded polypeptides of 387 amino acid residues.The molecular weight was 40 kD analysed by SDS-PAGE. The level of soluble expression product was about 41.67% of total cell protein. After affinity chromatography, the purity of fusion protein was above 92%.Western blot analysis confirmed that fusion protein could be specifically recognized by the serum of anti-HpaA and anti-CT. Antigen-specific ASC and antibody response in animals immunized with HCTB or HpaA was determined by ELISPOT and ELISA. The results showed that the number of sIgA and IgG-ASC increased significantly in PP and gastric mucosa, especially those of the sIgA-ASC by orally immunization with HCTB. The levels of specific antibody were also higher than those of controls. Conclusion:The results indicated that the oral immunization with HCTB induces effective mucosal immune respones and produced higher levels sIgA. The recombinant fusion protein HCTB can be used as an effective oral vaccine for prevention and treatment of infection of Hp.
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Objective:Researching physical-chemic and biologic speciality of VacA-HpaA IgY,which provide a solid experimental basis for the preparation of vaccine against H.pylori infection.Methods:The purified antigen of fusion protein VacA-HpaA was used to immunize hens and the VacA-HpaA IgY was extracted from the yolk by water dilution methods.(1)After the VacA-HpaA IgY was purified by deposition technique with ammonium sulfate,the purity of IgY was analyzed by SDS-PAGE,and protein content of IgY was analyzed by Bradford method.(2)The indirect ELISA was used to detect its heat-stability,acid-stability and pepsin-stability.(3)The specificity of VacA-HpaA IgY to the antigens was identified by Western blot.(4)MTT was applied to assay the neutralization of VacA-HpaA IgY to The cytotoxity of H.pylori.(5)The neutralization of IgY to AGS cells was observed with scanning electron microscopy(SEM).Results:(1)The purity of VacA-HpaA IgY was about 60%,and the protein content of IgY was 22g/L;(2)The IgY showed a good heat-stability,a favorable acid-stability,and a well ability of anti-pepsin digest;(3)Western blot exhibited the protein bands with molecular weight of 27 000 and 30 000.The titer of VacA-HpaA IgY to VacA and HpaA were 1∶3 200 and 1∶6 400;(4)A dose and time dependent correlation of the IgY to counteract the cytotoxic activity of VacA;(5)VacA-HpaA IgY could interrupt the adhesive attraction of H.pylori.Conclusion:The VacA-HpaA IgY has good stabilities and well biologic speciality,which imply that the IgY could be used to prepare treatmental antibody against H.pylori infection.