Expression and assembly of chimeric flagellins in Escherichia coli BL21(DE3) and Salmonella / 生物工程学报

Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1β secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.

Research progress on Salmonella Derby / 中国人兽共患病学报

Salmonella Derby is recognized as a major human food-borne pathogen causing food poisoning,septicaemia and other symptoms.Meanwhile,it can represent a severe threat to livestock breeding and health.The objective of this review is to summarize novel research progress on epidemic,genomics,pathogenic mechanism of Salmonella Derby for provide reference to related research.

Development of swine Salmonella vaccine / 中国人兽共患病学报

Salmonella is an important zoonosis.China is the largest pork consumer.Contaminated pork is the main source of Salmonella disease at home and abroad.There are many swine Salmonella vaccine in use in Europe.Now the current swine Salmonella vaccine is a live attenuated vaccine,Salmonella can reduce colonization in pigs,and can induce immune response efficiently after the two immunizations.The control effect is poor,at this stage of swine vaccine cross protection research,type of Salmonella and vaccine antigen are not the same.This paper reviews the research progress of swine Salmonella vaccine.

Construction and characterization of an attenuated recombinant Listeria monocytogenes vector vaccine delivering HPV16 E7 / 生物工程学报

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD₅₀) of LM4△hly::E7 strain was 3.863×10⁹ CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.

Relationship between interferon-α conformation and its anti-viral activity determined by circular dichroism and flow cytometry / 生物工程学报

The relationship between the conformation of interferon-α (IFN-α) and its anti-viral activity were analyzed by circular dichroism (CD) and flow cytometry (FCM) techniques. The recombinant human IFN-α (rIFN-α2b and rIFN-α2a) were used. CD spectra from 190 nm to 240 nm indicated that two the IFN-α showed stable secondary structure at 65 degrees C, but unstable when the temperature was above 65 degrees C, and the change was irreversible. FCM data of the anti-viral activity of IFN-α indicated that the change of its secondary structures partly weakened its anti-viral activity. The rIFN-α2b and rIFN-α2a showed the same phenomenon. These data indicated that the conformation of IFN-α is one of the factors to influence its anti-viral activity and the combination of CD and FCM is a good method to analyze the relationship between the conformation of protein drugs and their biological activities in single cell level.

Research progress on T3SS relative effector protein of Salmonella / 中国人兽共患病学报

Salmonella is a pathogenic bacteria to human and animals ,which can cause seriously complication and death . Salmonella pathogenicity is from the reactions of SP‐1 and SP‐2 T3SS effector proteins .In this paper ,the functions of different T3ss effector proteins in different infection periods is reviewed to provide a reference for further understanding the pathogenesis mechanisms of Salmonella .

Analysis of the Safety of Antibiotic Drug Use by Children Being Referred and Hospitalized under Medical Consortium Model / 中国药房

OBJECTIVE:To provide reference for safe application of antibiotic drugs in single or inter-medical institution ser-vices. METHODS:An antibiotic drug safety evaluation index system was established by reference to literatures. Medical records tracing method was adopted to obtain the medical records of children with respiratory infectious diseases under the medical consor-tium model. The medical records were matched and evaluated by experts to analysis the safety of antibiotic drug use in single medi-cal institution and the connection between two medical institutions with respect to the safety of antibiotic drug use. RESULTS:248 effective medical records had been obtained. Under the medical consortium model,the rate of the combination of antibiotic drugs in large hospitals was 86.29%,higher than the community hospitals of 77.02%(χ2=5.49,P<0.05). The error rate of antibiotic drug use by children in large hospitals was 14.52%,lower than the community hospitals of 25.81%(χ2=9.733,P<0.05). There were many contradictions in antibiotic drug use between the medical institution where children received treatment initially and the medi-cal institution which the children were referred to and hospitalized in. There were totally 128 cases of unsafe antibiotic drug use, with the overall incidence of 51.61%. CONCLUSIONS:Under the medical consortium model,the safety of antibiotic drug use by the hospitalized children in single medical institution is worrying,and the connection between two medical institutions with respect to the safety of antibiotic drug use by children who are referred and hospitalized is less reliable.

Immunological characteristics of Mycobacterium tuberculosis antigen Rv2628 / 生物工程学报

Antigen Rv2628 of Mycobacterium tuberculosis is associated with latent tuberculosis infection. In this study, Rv2628 was prokaryotic expressed and purified, its immunological characteristics was evaluated with macrophage cell line RAW264.7 and BALB/c mice. The results show that Rv2628 was mainly expressed in form of inclusion body confirmed by SDS-PAGE, and could react with rabbit anti-H37Rv polyclonal antibody detected by Western blotting assay, indicating that the protein had an effective immunoreactivity. The interactions between Rv2628 and macrophage cell line RAW264.7 confirmed that it could effectively induce cells to produce pro-inflammatory cytokines, the relative expression level of IL-6 mRNA was higher than the control group in 1-12 h. BALB/c mice were subcutaneously immunized with Rv2628 protein, the production of IFN-gamma and IL-4 in the spleen cells was determined by Sandwich ELISA, in the Rv2628 immunized group, the level of IFN-gamma was significantly higher than that of IL-4 (P < 0.000 1). It indicated the protein induced Th1-tendency immune responses. At the same time, Rv2628(11-30) peptide used as coating antigen, the murine serum antibody titer detected by indirect-ELISA was 1:1 600, which demonstrated that Rv2628 could also induce humoral immune responses. In summary, Rv2628 could induce specific pro-inflammatory cytokines, affectively induce strongly Th1-tendency immune response and humoral response, it could be a potential target for developing subunit vaccine against TB. In addition, it laid foundation for probing the cross-talk between M. tb and host.

Progress of Listeria monocy togenes as tumor vaccine vector / 中国人兽共患病学报

Listeriamonocytogenes is a facultative intracellular bacterium that enters professional antigen presenting cells , presents passenger antigens to the major histocompatibility complex class I and II pathways ,then elicits CD+4 and CD+8 T-cell-mediated immune responses .It was demonstrated that attenuated Listeriamonocytogenes as a novel live vaccine vector in deliv-ering tumor antigens of cervical cancer and melanoma etc .,could induce strong protective immune response ,and shows effec-tive antitumor immunotherapeutics .This review discussed the characteristics of immune responses elicited by Listeria monocy-togenes ,and the progress of its antitumor immunotherapeutics as delivery vaccine vector .

Epidemiology of listeriosis infection in humans and animals, China, 2002-2012 / 中国人兽共患病学报

The prevalence of human and animal listeriosis for nearly 11 years in China was investigated in this study . The literature information about listeriosis in China from 2002 to 2012 was collected through retrieval system to make clinical and epidemiological statistical analysis of listeriosis .Cases of listeriosis were reported in 27 (79% ) provinces of China .The re-sult showed that animal listeriosis was reported for 123 times ,among these reports ,most were from pigs (39% ) ,and the sheep was in second place .Central nervous system infection was the main clinical manifestation of listeriosis in animals (72% ) . For human listeriosis ,84 clinical cases of listeriosis were reported ,including 35% cases in non-perinatal stage and 65% cases in perinatal stage .The main clinical manifestation of listeriosis was septicemia (51% ) .According to the result of investigation about listeriosis based on literatures information ,Listeriamonocytogenes caused humans and animals listeriosis annually ,which were reported in most provinces of China .The epidemic characteristics for listeriosis suggested that it was essential to strength-en the prevention and control of listeriosis .

Expression and antiviral assay of bovine interferon-gamma / 生物工程学报

Bovine interferon-gamma (BoIFN-gamma) gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes stimulated with ConA. The products of RT-PCR were cloned into pVAX1 vector, positive recombinant clone was identified by restriction enzyme digestion and sequencing. The recombinant plasmid pVAX1-BolFN-gamma was transfected into COS-7 cells mediated by lipofectine, indirect immunofluorescent assay analysis confirmed that rBoIFN-gamma was expressed in COS-7 cells. BoIFN-gamma gene (without signal peptide) was cloned into pET-30a(+) and pGEX-6p-1 vector, and transformed into the Escherichia coli cells. After optimizing the induction condition, SDS-PAGE analysis showed that the expression products were all found in soluble form and had a molecular weight of 23 kDa and 43 kDa respectively. BoLFN-gamma precursor gene (with signal peptide) was cloned into transfer vector pFastBac 1, and transformated into DH10Bac E. coli cells. By site-specific transposition, BoIFN-gamma gene was integrated into shuttle vector Bacmid, and transfected into the Sf9 insect cells mediated by lipofectine to produce recombinant baculovirus. Indirect immunofluorescent assay analysis confirmed that rBac-BoLFN-gamma was expressed successfully in Baculovirus vector system. The antiviral activities of rHis-BoIFN-gamma, rGST-BoIFN-gamma and rBac-BoIFN-gamma were up to 8.389 x10(7) U/mg, 6.554 x10(5) U/mg and 4.096 x 10(4) U/mL respectively, which were analyzed in MDBK/VSV system. A sandwich ELISA was established using monoclonal antibodies 3E6 and 5G4, which can detect BoIFN-gamma in quantity and provide a useful method for the clinical practice and research of BolFN-gamma.

Immune responses specific for ESAT-6 expressed by recombinant Salmonella typhimurium / 中华微生物学和免疫学杂志

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.

The analysis of quinolone resistance of the avian Escherichia coli and Salmonella isolates from 1993 to 2008 / 中国人兽共患病学报

Quinolones are broad-spectrum antibacterial agents used in human and veterinary medicine, and their extensive use have been associated with a rise of the quinolone resistance. In the present study, the quinolone resistance of avian E.coli and Salmonella isolates was evaluated and compared, in which 344 avian E.coli and 224 Salmonella isolates from 1990s were serogrouped with antisera and thc antimicrobial susceptibility test to 10 quinolones was carried out by using the Kirby-Bauer method recommended by Clinical and Laboratory Standards Institute (CLSI). It was demonstrated that the 344 isolates of avian E.coli distributed in 27 serogroups and 68.90% (237/344) of the isolates belonged to four O-serogroups: i.e. O1, O2, O18, O78, and the 224 isolates of avian Salmonella were all determined to be Salmonella pullorum. The drug-resistance rate of avian E. coli isolates to nalicixic acid from 1993-1999 was more than 60%(64.43%,131/181), whereas those of isolates to 9 antibiotics from 2000-2008 had a drug-resistance rates of more than 60%, namely,nalicixic acid(92.02%), fleroxacin(79.75%), pipemidic acid(79.14%), enrofloxacin(78.53%), enoxacin(76.07%), lomenfloxacin(74.85%), ciprofloxacin(69.33%), norfloxacin(63.80%) and ofloxacin(61.35%). For the 4 O-serogroups of the avian E.coli isolates, the drug-resistance rates of more than 50% to antimicrobials were as follows: O78 isolates to 7 antimicrobials;O18 isolates to 5 antimicrobials, and O1 and O2 isolates just to 3 antimicrobials. The quinolone resistance of Salmonella isolates was much lower than E.coli, in which 101 salmonella isolates from 1993-1999 were all susceptible to quinolones. Nalicixic acid resistance of salmonella isolate firstly appeared in 2000, and the drug-resistance rate of salmonella isolates from 2000-2008 was found to be more than 60% for nalicixic acid(83.74%), but those to other quinolones were comparatively lower. These results indicated that the quinolone resistance of avian E.coli and salmonella were increasing in the past two decads because of the over-use of antibiotics.

Construction and characterization of avian pathogenic Escherichia coli mutants with iro and/or tsh gene mutation / 生物工程学报

Iro system and temperature-sensitive hemagglutinin (Tsh) genes were identified by suppression subtractive hybridization (SSH) and selective capture of transcribed sequences (SCOTS). To get more insights in the distribution and the occurrence of the iroC and tsh genes, we examined 243 avian E. coli strains for the presences of the these genes. Among 243 avian E. coli isolates, iroC gene was present in 84.4% strains (205/243). Of the 205 iroC-positive isolates, iroC gene was found in 184 (89.8%), 18(8.8%) and 3 (1.5%) isolates with high, intermediate and low pathogenicity, respectively. Of the 167 tsh-positive isolates, tsh gene was detected in 146 (87.4%), 21 (12.6%) and 0 (0%) isolates with high, intermediate and low pathogenicity, respectively. Among tsh-positive isolates, 89.5 to 100% of the highly pathogenic isolates of O1, O2 or O78 serogroups had the tsh gene, while 53.3% of the highly pathogenic isolates of non-O1, O2 and O78 serogroups had the tsh gene (P<0.01). Suicide vectors for deletion of the iroBCDEN or tsh genes were constructed as follows. The 715-bp fragments of iroB and 603-bp fragment of the iroN were generated by PCR respectively. Both of these two fragments together with EGFP gene were cloned into pUC18, termed pUC18-iroBNEGFP. A resultant suicide vector containing the iroB-EGFP-iroN fragment was obtained and named pMEG375-iroBNEGFP. Similarly, both of the 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18, resulting in pUC18-tshFRGm. A resultant suicide vector containing the tshF-Gm-tshR fragment was named pMEG375-tshFRGm. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037(Deltairo), E037(Deltatsh) and E037(DeltairoDeltatsh). The 50% lethal dose (LD50) of E037, E037(Deltairo), E037(Deltatsh) and E037(DeltairoDeltatsh) in commercial day-old chickens experimentally inoculated via intratrachea were determined to be 10(5.6), 10(8.4), 10(9.0) and 10(9.5)CFU, respectively. In the chicken challenging model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. The result suggested that Iro system and Tsh were important in the pathogenicity of APEC.

Study on CD8~+ T cell epitope expressed by eukaryotic plasmid delivered by attenuated E.coli / 中国免疫学杂志

Objective:To analyze the efficiency of delivery for CD8+ T cell epitope by attenuated E.coli vector.Methods:The recombinant E.coli strain 13A(pG2F),harbouring the eukaryotic expression plasmid pG2F with CD8+ T cell epitope of Ovalbumin (OVA) and green fluorescent protein (GFP) marker at the C-terminal was used to infect into LKb cells,bone marrow dendritic cells (BMDC).The efficiency of presentation for CD8+T cell epitope delivered by recombinant bacteria was analyzed by in vitro antigen presentation assay.C57BL/6 mice were immunized intravenously with 13A(pG2F).Murine IFN-? secreting cells were detected in murine splenocytes by enzyme-linked immunospot assay (ELISPOT).Results:After the infection of LKb cells,BMDC by recombinant bacteria,about 0.3%～4% of cells were GFP positive.The results indicated that attenuated strain 13A could deliver the eukaryotic expression plasmid into mammalian cells.At 2 h post infection,CD8+ T cell epitope was presented on the surface of those LKb,BMDC cells infected by 13A (pG2F) could be recognized by B3Z T hybridoma cells.The presentation efficiency of LKb cells for OVA CD8+ T cell epitope was increased at 48h after infection.Furthermore,the presentation efficiency of BMDC was higher than those of LKb cells under the same condition.The recombinant bacteria 13A(pG2F) could induce cellular immune responses in C57BL/6 mice.Conclusion:Attenuated E.coli can effectively deliver the CD8+ T cell epitope in vitro and in vivo.