• 대한수의학회지
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• 제46권2호
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• pp.127-133
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• 2006

Pasteurella multocida is a terrible veterinary pathogen that causes widespread infections in husbandry. To induce homologous and/or heterologous immunity against the infections, outer membrane protein Hs (OmpH) in the envelope of different strains of P. multocida are thought to be attractive vaccine candidates. Previously we cloned and characterized a gene for OmpH from pathogenic P. multocida (A : 3) (In Press, Korean J. Microbiol. Biotechnol. 2005, 33, December). The gene is composed of 1,047 nucleotides (nt) coding 348 amino acids (aa) with signal peptide of 20 aa. The truncated ompH, a gene without nt coding for the signal peptide, was generated using pRSET A to name "pRSET A/OmpH-F2". This truncated ompH was well expressed in Escherichia coli BL21 (DE3). Truncated OmpH was purified for induction of immunity against live pathogen of fowl cholera (P. multocida A : 3) in mice. Some $50{\mu}g$ of the purified polypeptide was intraperitoneally injected into mice two times with 10 day interval. Lethal dose ($25{\mu}l$) of live P. multocida A : 3 was determined by directly injecting the pathogen into wild mice (n = 25). To demonstrate the vaccine candidate of the truncated OmpH, the live pathogen ($25{\mu}l$) was challenged with the OmpH-immunized mouse group as well as positive & negative controls (n = 80). The results show that the truncated OmpH can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (A : 3).

• Parasites, Hosts and Diseases
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• 제47권4호
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• pp.359-367
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• 2009

Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis.

• BMB Reports
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• 제40권6호
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• Tuberculosis and Respiratory Diseases
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• 제57권2호
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• pp.125-131
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• 2004

결핵균 감염에 대한 주요 방어 항원 물질로서 항원 85 복합체(Ag85A, B, C)가 주목되고 있다. 우리는 이들 항원들을 과발현하는 재조합 BCG를 클로닝하였고, 마우스 모델을 이용하여 결핵균 감염에 대한 재조합 BCG의 방어 효능을 알아보고자 하였다. 항원 85A를 과발현하는 재조합 BCG를 rBCG/FA, 항원 85B를 과발현하는 재조합 BCG를 rBCG/FB, 그리고, 이들 두 항원을 과발현하는 재조합 BCG를 rBCG/B.FA라고 명명하였고, 이들 항원들의 과발현 여부를 SDSPAGE상에서 확인한 결과, 재조합 BCG에서 항원 85A와 B 단백질이 BCG에 비해 과발현된 것을 알 수 있었다. 면역주사한 마우스에서 분리한 비장세포를 M. tuberculosis H37Rv의 culture filtrate protein(CFP)으로 자극하여 분비된 IFN-${\gamma}$ 농도를 측정한 결과에서는 rBCG/B.FA만이 BCG에 비해 높은 IFN-${\gamma}$ 농도를 나타내었으나, 마우스 모델을 이용한 결핵균 감염에 대한 재조합 BCG의 방어 효능 실험에서는 BCG와 뚜렷한 차이를 나타내지 못하였다.

• Journal of Microbiology and Biotechnology
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• 제27권9호
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• pp.1701-1710
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• 2017

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that $RecCpp40_{+10}$ failed to induce fever in rabbits, whereas $RecCpp80_{+10}$ caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.330-338
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• 2019

Chronic infection with intracellular Brucella abortus (B. abortus) in livestock remains as a major problem worldwide. Thus, the search for an ideal vaccine is still ongoing. In this study, we evaluated the protective efficacy of a combination of B. abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12) and malate dehydrogenase (rMDH), cloned and expressed into a pMal vector system and $DH5{\alpha}$, respectively, and further purified and applied intraperitoneally into BALB/c mice. After first immunization and two boosters, mice were infected intraperitoneally (IP) with $5{\times}10^4CFU$ of virulent B. abortus 544. Spleens were harvested and bacterial loads were evaluated at two weeks post-infection. Results revealed that this combination showed significant reduction in bacterial colonization in the spleen with a log protection unit of 1.31, which is comparable to the average protection conferred by the widely used live attenuated vaccine RB51. Cytokine analysis exhibited enhancement of cell-mediated immune response as IFN-${\gamma}$ is significantly elevated while IL-10, which is considered beneficial to the pathogen's survival, was reduced compared to control group. Furthermore, both titers of IgG1 and IgG2a were significantly elevated at three and four-week time points from first immunization. In summary, our in vivo data revealed that vaccination with a combination of five different proteins conferred a heightened host response to Brucella infection through cell-mediated immunity which is desirable in the control of intracellular pathogens. Thus, this combination might be considered for further improvement as a potential candidate vaccine against Brucella infection.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2095-2105
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• 2018

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

• IMMUNE NETWORK
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• 제13권4호
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• pp.157-162
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• 2013

Application of vaccine materials through oral mucosal route confers great economical advantage in animal farming industry due to much less vaccination cost compared with that of injection-based vaccination. In particular, oral administration of recombinant protein antigen against foot-and- mouth disease virus (FMDV) is an ideal strategy because it is safe from FMDV transmission during vaccine production and can induce antigen-specific immune response in mucosal compartments, where FMDV infection has been initiated, which is hardly achievable through parenteral immunization. Given that effective delivery of vaccine materials into immune inductive sites is prerequisite for effective oral mucosal vaccination, M cell-targeting strategy is crucial in successful vaccination since M cells are main gateway for luminal antigen influx into mucosal lymphoid tissue. Here, we applied previously identified M cell-targeting ligand Co1 to VP1 of FMDV in order to test the possible oral mucosal vaccination against FMDV infection. M cell-targeting ligand Co1-conjugated VP1 interacted efficiently with M cells of Peyer's patch. In addition, oral administration of ligand-conjugated VP1 enhanced the induction of VP1-specific IgG and IgA responses in systemic and mucosal compartments, respectively, in comparison with those from oral administration of VP1 alone. In addition, the enhanced VP1-specific immune response was found to be due to antigen-specific Th2-type cytokine production. Collectively, it is suggested that the M cell-targeting strategy could be applied to develop efficient oral mucosal vaccine against FMDV infection.

• 대한수의학회지
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• 제57권3호
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• pp.155-158
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• 2017

Surgical castration performed to reduce male-associated problems results in pain and microbial infections in male animals. Therefore, immunocontraception, which is mediated by the animal's own antibodies against reproductive hormones, has been recommended as an alternative to surgical castration when considering the animal's welfare. In this study, a new immunocontraceptive vaccine composed of six tandem copies of gonadotropin-releasing hormone (GnRH) fused to rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was developed, and its efficacy was evaluated in male rats. Three different doses (10, 50, and $100{\mu}g$) of recombinant GM-CSF-GnRH protein were injected three times at intervals of two weeks into male rats. The rats vaccinated with three doses of GM-CSF-GnRH produced a significantly higher level of antibodies against GnRH than that in the negative control rats. Severe atrophy of gonads was observed in rats vaccinated with three doses of GM-CSF-GnRH but not in the negative control rats. The results reveal that the new GnRH vaccine conjugated with rat GM-CSF induces efficient immunocontraception in male rats. This formulation of the immunocontraceptive vaccine would be applicable to both domestic and pet male animals.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.318-324
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• 2006

In order to develop an oral vaccine to prevent H. pylori infection, we have expressed the hpaA gene of H. pylori K51 isolated from Korean patients, encoding 29-kDa HpaA that is known to be localized on the cell surface and flagella sheath, in a live delivery vector system, Lactococcus lactis. The hpaA gene, amplified by PCR using the genomic DNA of H. pylori K51, was cloned in the pGEX-2T vector, and the DNA sequence analysis revealed that the hpaA gene of H. pylori K51 had 99.7% and 94.8% identity with individual hpaA genes of the H. pylori 26695 strain (U.K) and the J99 strain (U.S.A). A polyclonal anti-HpaA antibody was raised in rats using GST-HpaA fusion protein as the antigen. The hpaA gene was inserted in an E. coli-L. lactis-shuttle vector (pMG36e) to express in L. lactis. Western blot analysis showed that the expression level of HpaA in the L. lactis transformant remained constant from the exponential phase to the stationary phase, without extracelluar secretion. These results indicate that the HpaA of H. pylori K51 was successfully expressed in L. lactis, and suggest that the recombinant L. lactis expressing HpaA may be applicable as an oral vaccine to induce a protective immune response against H. pylori.