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Objective To prepare monoclonal antibodies (mAb) against the type Ⅳ secretion system protein VirB5 of Brucella melitensis and to provide a basis for pathgenic diagnosis and research of brucellosis.Methods Four SPF female BALB/c mice were subcutaneously immunized with purified VirB5 protein at a dose of 60 μg/mice,and immunization was strengthened every 2 weeks at a dose of 30 μg/mice,three times in total.Two weeks later,the orbital venous blood of mice was taken to determine the antibody titer,and then intraperitoneally injected for the fourth time to strengthen immunization.Three days later,mouse spleen cells were fused with mouse myeloma SP2/O cells in a ratio of 5:1.After 3 times of cell screening and monoclonal cloning,the hybridoma cell lines with stable secretion of VirB5 antibody were established;one BALB/c mouse was intraperitoneally injected with hybridoma cells,and ascites were collected and antibody was purified when the mouse abdomen was significantly enlarged.The immunological characteristics of mAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting.Results A total of 6 monoclonal cell lines (2-2,2-12,2-19,2-25,2-31 and 2-40) capable of secreting VirB5 antibody were established.Among them,the cell line 2-19 can stably secrete an antibody that specifically recognized the VirB5 protein,and the VirB5 antibody secreted by the cell line was identified as an IgG1 subtype,a kappa light chain,a mAb affinity constant of 1.6 × 108.The titer of ascites antibody of mouse intraperitoneally injected with hybridoma cell 2-19 was 1:51 200.Conclusion The high-affinity mAb of type Ⅳ secretion system protein VirB5 is successfully prepared,and the antibody can rapidly bind specifically to pathogens,providing an alternative material for establishment of brucellosis pathogen diagnostic method.
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Objective@#To investigate the function of virD4 gene in Helicobacter pylori clinical strain SBK. @*Methods@#The virD4 gene segment was obtained through T-A cloning method. The prokaryotic expression vector pET-28a(＋)-virD4 was constructed and transformed into E. coli Rosetta for the expression by induction of IPTG. The recombinant proteins were obtained and purified by KCl dyeing with gel cutting method, and identified via SDS-PAGE analysis. The purified recombinant virD4 protein was used to immunize mice to produce polyclonal antibodies. The titer of the polyclonal antibodies was tested by ELISA and the antigenic specificity was identified by western blot. The purified recombinant virD4 proteins were co-cultured with GES-1 cells followed by detecting the expression of inflammatory cytokines secretion and the ability of cell proliferation. @*Results@#The full length of virD4 gene was 1 728 bp. The sequence shared quite high homology with the virD4 gene of isolate Shi470. The recombinant prokaryotic expression plasmid pET-28a(＋)-virD4 was successfully constructed. The recombinant virD4 proteins were obtained by IPTG induction and purified via KCl dyeing method. SDS-PAGE showed that the relative molecular weight of recombinant virD4 protein was 63 000. The purified proteins were used to immunize mice to obtain the anti-virD4 polyclonal antibodies with the titer 512 000. The reaction between anti-virD4 polyclonal antibodies and recombinant virD4 proteins was highly specific. The recombinant virD4 protein induced inflammatory cytokines secretion and promoted GES-1 cell proliferation. @*Conclusion@#The virD4 gene was successfully cloned and highly expressed in prokaryotic expression system, and its antibodies were prepared. The recombinant virD4 protein can induce cytokine secretion and cell proliferation.
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Objective To analyze the sequence of the novel conjugative plasmid pO157_Sal detected in outbreak isolates of Escherichia coli O157∶H7 .Methods The traE genes of the outbreak isolates in China were amplified by polymerase chain reaction (PCR) and the products were sequenced .The TraE sequences of Escherichia coli O157 ∶ H7 strains from other sources were retrieved from GenBank . Phylogenetic tree based on the TraE sequences was constructed by Neibhor-joining analysis .The whole plasmid sequences of pO157_Sal and pEC4115 were compared .Results The sequences of traE gene were identical among the Chinese isolates . There were homologous sequences of TraE in Escherichia coli O157∶H7 isolates from different sources .Twenty-one out of the 52 pO157_Sal genes were homologous to genes of pEC4115 with amino acid level identity ranging from 28% to 51% .Conclusions Although similar TraE sequences and similar plasmid are found in Escherichia coli O157∶H7 isolates from different sources ,pO157_Sal is only observed in Chinese outbreak isolates .The TraE sequences are conservative among the outbreak isolates ,indicating they are from the same specific source .
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Objective To detect the immunogenicity of VirB9,a protein of type Ⅳ secretion system of Brucella.Methods Full length VirB9 gene was cloned into plasmid pET32a and expressed in Escherichia (E.) coli BL21 (DE3).Expression of recombinant protein was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and the recombinant fusion protein was purified by affinity chromatography on Ni2+-conjugated chelateing sepharose.The purity of the purified protein was ascertained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE) and the concentration was measured by bicinchoninic acid (BCA) protein assay kit.Animal model was established by immunizing BAL B/c mice with live vaccine strain S19 of Brucella and the mice immunized with phosphate buffered saline (PBS) as control.The blood of immunized mice was acquired after 4 weeks.Antibody against VirB9 in S19 immunized mice was detected by Rose Bengal plate agglutination test and serum tube agglutination test; IgG antibody titers against VirB9 in immunized mice were determined by enzyme linked immunosorbent assay(ELISA).At the 35th day,the immunized mice and control mice were killed and spleens were collected.The splenocytes were harvested and stimulated with each of VirB9,concanvalin A(ConA) or medium in triplicate.Production of gamma interferon (IFN-γ) was determined by enzyme-linked immunospot assay (Elispot).Results The full length of VirB9 gene was cloned into pET32a.The recombinant VirB9 protein was expressed at 43 × 103 in relative molecular mass and the purity of the purified recombinant VirB9 protein was above 97％ in SDS-PAGE and the concentration was 1.6 g/L in BCA protein assay.The antibody of VirB9 was detected in all S19 immunized mice but not PBS immunized mice by Rose Bengal plate agglutination test.The antibody titer in all S19 immunized mice was ＞ 1 ∶ 800 or ＞ 1 ∶ 3200 by tube agglutination test and ELISA,respectively.Meanwhile,the protein stimulated stronger IFN-γresponse in immunized mice than that in the control mice(147 cells Vs 38 cells).Conclusion VirB9 can stimulate humoral and cellular immunity and it might be an appropriate target for developing subunit vaccine against Brucella.
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Agrobacterium tumefaciens can transfer a DNA fragment (T-DNA) from its Ti plasmid to host plant and integrate the T-DNA fragment into host cell nuclear genome. Agrobacterium-mediated T-DNA transfer is the most widely used genetic transformation method for plant. The T-DNA is delivered in the form of single-stranded T-DNA-protein complex (T-complex) by the polar-located Agrobacterium type Ⅳ secretion system (T4SS) to the host cell. T4SS is ancestrally related to bacterial conjugation machines and still used by many plasmids as conjugation channel. Moreover, T4SS is also the secretion channel that used by many human bacterial pathogens to inject the effector proteins to host cells, thus contributing directly to the bacterial pathogenicity. Therefore, in addition to the technical application as a gene vector to create transgenic plants, Agrobacterium-mediated T-DNA transfer also provides a fascinating model system to study the intercellular transfer of macromolecules. The study on the molecular mechanism of T-DNA transfer arouses extensive interest and progresses rapidly in recent years. Here the recent advances in research on T-complex formation and T-complex transfer in Agrobacterium cell are reviewed.