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Bacteria play an important role in human health and disease. Their biological functions are often related to the glycans attached to the protein surface. In recent years, the glycosylation modification of bacterial proteins has attracted increasingly widespread attention. With the continuous development of synthetic biology and the in-depth research on glycosylation modification systems, some modification systems have been applied in engineered bacteria to play the role of protein modification independently, making it possible to "customized glycoproteins" . This paper reviewed the current status of research on the basic components, types and pathways of bacterial protein glycosylation modification as well as the biological function and application.
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The research of structural biology is closely related to the field of biotechnology.Carrying out the second class of structural biology will be helpful in learning theoretical knowledge of biotechnology and in improving the teaching quality.Based on our experiences of carrying out the second classroom activities among students majoring in biotechnology,we believe that the selected teaching contents,reasonable classroom design,sufficient preparation before the class,flexible teaching methods and objective after-school summaries are essential for improving the teaching quality.
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Objective To analyze the combination characteristics between Tir-IBD( intimin binding domain) and its ligand intimin or mutant intiminN916Y of EHEC O157 ∶H7.Methods The gene of TirIBD (tir-ibd) from EHEC O157 ∶H7 chromosome was cloned into pMD18-T vector.Thereafter,the amplified gene was cloned into prokaryotic expression plasmid pET-21 a (+).The recombinant pasmid pET-21 a( +)-tir-ibd was transformed into E.coli BL21 (DE3).After inducement,the protein Tir-IBD was successfully expressed and analyzed with SDS-PAGE and Western blot.It was purified by affinity chromatography and ionexchange chromatography and was coupled on the Ni-NTA chip of BIACore 3000.Then the ligand intimin and mutant intiminN916Y were flow through the chip and their combination characteristics were detected.Results In the present study,the gene of Tir-IBD(tir-ibd) was successfully cloned into pET-21a(+).The results of SDS-PAGE and Western blot assay showed that the protein was successfully expressed,which accounts for 15％ of total expression products,and its molecular weight was about 10×103.The purity was above 95％ after purification.After coupled on the Ni-NTA chip of BIACore 3000,their combination characteristics with ligand intimin and mutant intiminN916Y were successfully detected.The equilibrium binding constants Ka was obtained by fitting the data with the BIACore evaluation program ( Version 4.1 ).The result showed that the combination characteristics between Tir-IBD and intimin have some difference compared with that of mutant intiminN916Y and the difference is temperature dependent.Conclusion Tir-IBD of EHEC O157 ∶H7 was successfully constructed and purified.The method to analyze the combination characteristics between Tir-IBD and its ligand intimin or mutant was established.The combination characteristics between Tir-IBD and intimin or mutant intiminN916Y have some temperature dependent difference and the mutated amino acids residue is crucial for their receptor-ligand binding.
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Objective To clone the gene encoding protein of EspA and Stx2B from EHEC OI57:H7 by DNA recombinant technology, construct prokaryotic expression vector pET-28a ( + )-espAstx2B, express fusion protein of EspA-Stx2B and to analyze the biological and immunological characteristics of the fusion protein. Methods the sequence encoding the protein of EspA and Stx2B was amplified by PCR from the enterohemorrhagic Escherichia coli strain. The amplified products were connected with linker by recombinant technology and cloned into pET-28a( + ) vector. The vector was then transferred to the host cells E. Coli BL21 strain (DE3). Following, the protein expression was induced by IPTG. The expression quantities and style of fusion protein was then determined by SDS-PAGE. Its immunoreactivity was analyzed by Western blot. Finally, BALB/c mice were injected with the preliminarily purified recombination protein EspA-Stx2B, then oral challenged these mice with EHEC O157-SMR2 and counteracted toxic substances with O157 ultrasonic supernatant. Results The determination of the sequence encoding of the espA-stx2B fusion gene has 100% of consistency with the sequence from GenBank Sakai strain and contrivable linker. This fusion protein EspA-Stx2B was expressed as inclusion body formation and the percentage is approximately 40%. Western blot suggested the fusion protein has excellent immunoreactivity. Titer of antiserum of the mice to EspA-Stx2B increased evidently. EspA-Stx2B could not decrease bacterial number attached to the intestinal tract of mice based on fecal shedding of Oi57 in mice. In the test of death of BAI,B/c causing by conteracting toxic substances with O157 ultrasonic supernatant, immunoprotection of EspA-Stx2B rate was 66.7%. Conclusion A recombinant plasmid that has high performance on expression of EspA-Stx2B prorein was successfully constructed in present study, and the fusion protein has excellent immunoreactivity and immunogenicity. EspA-Stx2B could not decrease bacteria] number attached to the intestinal tract of mice based on fecal shedding of O157 in mice, but evidently decrease the mortality rate of the mice. The antiEspA and anti-Stx2B had immunoprotection effect by different means. These results may provide the foundation for the further development on EHEC O157:H7 double subunit vaccine.
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Objective To investigate immunoprophylactic potential of genetic engineering vaccines of enterohaemorrhagie Escherichia coli O157:H7 in BALB/c mice after immunization with these vaccines. Methods Sixty BALB/c mice (3 weeks old) were randomized averagely into 5 groups. Group 1-3 were im-munized respectively with IntiminC300, Stx2B and HIyAN436, group 4 with a combination of these three vaccines, and group 5 with PBS. Each mouse was immunized with vaccine(100 μg)and Al(OH)3 adjuvant (100 μg) for 3 times. After 7 d of the second and third immunization, serum of each mouse was collected and the different antibodies were detected. After 10 d of the last immunization, all mice were given drinking water containing streptomycin for 3 d before and following oral challenge with O157:H7 (109 CFU), and treated with clinical, microbiological and pathological examination. Results The three vaccines elicited high titer antiserum, and some mice were died after infection with O157. The livability of group 1-4 was re-spectively 73%, 64%, 36% and 91%. And these vaccines depressed fecal and colon shedding with O157. Condusion IntiminC300, Stx2B and HIyAN436 have certain protective efficacy for infection of O157, and combined immunization was more effective than single vaccine.
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<p><b>OBJECTIVE</b>To express a fusion protein of Neisseria gonorrhoeae with a mucosal adjuvant.</p><p><b>METHODS</b>The gene coding Loop VI-VIII(PL678) of porin, an out-membrane protein of Neisseria gonorrhoeae, was obtained by PCR. It was inserted into a plasmid fused with subunit B of heat labile enterotoxin. The recombinant was transformed in E. coli. The expression of fusion protein was analysed by ELISA, SDS-PAGE and Western-blot.</p><p><b>RESULT</b>Fusion protein with LTB was successfully expressed, and displayed both the ability of binding GM1 and the reactogenicity with polyclonal antibodies against Neisseria gonorrhoeae.</p><p><b>CONCLUSION</b>The expression of fusion protein laid a foundation for the study of the intramolecular vaccine against Neisseria gonorrhoeae.</p>
Subject(s)
Bacterial Outer Membrane Proteins , Bacterial Toxins , Bacterial Vaccines , Allergy and Immunology , Enterotoxins , Escherichia coli , Genetics , Escherichia coli Proteins , Neisseria gonorrhoeae , Chemistry , Allergy and Immunology , Polymerase Chain Reaction , Recombinant Fusion Proteins , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
Objective To construct a fusion expression vector with subunit B of Escherichia coli heat labile enterotoxin. Methods Gene encoding LTB without stop codon obtained by PCR was introduced to a linker and was recombined on vector Pinpoint Xa I through Eco RⅠ and Eco RⅤ sites and then fused with ureB. The recombinant was used to transform E. coli JM109. The expression of LTB was analyzed by SDS PAGE and Western blotting. Results The fusion expression plasmid was successfully constructed. The subunit B of urease of Helicobacter pylori (Hp) fusogenic protein with LTB with the ability to bind GM 1 and the reactogenicity with polyclonal antibodies against Hp was harvested. Conclusion The successfully constructed vector provides experimental base for the studies of intramolecular adjuvant vaccine.
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MicroRNAs (miRNA) are a newly discovered class of endogenous,evolutionarily conserved small noncoding RNAs involved in posttranscriptional gene regulation. Functionally speaking,miRNAs act as key regulators in a wide variety of biological processes,including cell proliferation,cell differentiation,apoptosis,metabolism,developmental timing,signal transduction and tumor. Recent publications have provided compelling evidence that a range of miRNAs are involved in the regulation of immunity,including the innate immunity,proliferation of monocytes and neutrophils,the development and differentiation of B and T cells,infection and immunity,and the release of inflammatory mediators. In this review,we examine what is presently known of the function and mechanism of these miRNAs in the regulation of the innate and acquired immune response.
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Objective To prepare high-titer monoclonal antibodies against STX2A1 subunit of enterohemorrhagic E.coli(EHEC) O157∶H7.Methods BALB/c mice were immunized with GST-STX2A1 fusion protein and the spleen cells of BALB/c mice which were not immunized were used as feeder cells.Hybridoma technique,natural STX2A protein and ELISA test were used to prepare and screen the hybridoma cell lines of monoclonal antibodies against STX2A1.The ascites developed by injecting the hybridoma cells into abdominal cavity of the BALB/c mice and was purified with Protein A-Sepharose.The subclasses and isotypes were identified by mouse monoclonal antibody isotyping kit.The antigenic epitopes that can be recognized by STX2-1A3,STX2-1E10 and STX2-3A7 were analyzed by the ELISA additivity test.Results Three hybridoma cell strains were obtained and named as STX2-1A3,STX2-1E10 and STX2-3A7,respectively,all of which produced monoclonal antibodies specifically against STX2A1.The isotypes of the monoclonal antibodies were IgG1?,IgG1?,and IgG3? and the affinity constant was 5.76 ?109,1.21 ?109 and 3.97 ?108,respectively.Conclusion We have successfully prepared three hybridoma cell strains which secrete high-titer and highly specific monoclonal antibodies against STX2A1.Our study provides a basis for researching the early diagnosis,prevention and cure of the disease induced by EHEC O157∶H7.
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Objective To recombine and express a component of urease B subunit transmembrane protein of helicobacter pylori. Methods A 732 bp gene fragment of urease B subunit of helicobacter pylori was cloned into pET11C and transformed into BL21（DE3）E.coli. The positive clone was induced with IPTG. The expression of target protein was analysed by SDS-PAGE and Western blot. Results It is successful to construct the recombinant plasmid pET-UreB0.7 containing urease B subunit 0.7 kb gene fragment. A protein （MW≈28 000 u） with immunoreactivity， was expressed by 19.8％ in BL21（DE3）E.coli induced with IPTG. Conclusions The recombinant component of urease B subunit transmembrane protein may play a role in the research of its biological function and might be used as the vaccine against helicobacter pylori.
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To assess the variability of adhesin gene hpaA in differene H pyloristrains with PCR restriction fragment length polymorphism (RFLP) A 710 bp gene hpaA , obtained from 9 different H pylori strains, were digested by Hha Ⅰand Hae Ⅲ individually and analyzed by agarose gel electrophoresis Four different polymorphic types were found in hpaA digested with Hae III and five types with Hha I Clinical isolates of H pylori from Chongqing showed difference among them and remarkably distinguished from foreign standard strains Mongolia gerbil adapted H pylori strain,which were obtained from Mongolia gerbil infected with clinical isolate, also showed inconsistence in hpaA RFLP The hpaA gene from different H pylori strains revealed 1 variability, and this might provide an effective method for developing molecular epidemiology of H pylori