• International Journal of Industrial Entomology and Biomaterials
• /
• v.35 no.2
• /
• pp.118-122
• /
• 2017

Porcine pseudorabies virus (PRV) causes the Aujeszky's disease (AD) which is economically important disease in the swine industry worldwide. Killed or live vaccines have been used to control this disease, but their efficacy and side effects remain problems to be solved. To solve these problems, in this study, production of recombinant PRV glycoprotein gB, gC and gD was investigated in insect expression system. Glycoprotein gB, gC and gD are regarded as the major immunogenic antigens in PRV. Abundant production and immunogenicity of glycoprotein gB, gC and gD were confirmed by SDS-PAGE and Western blot analysis, respectively. Optimal infection dose and time were also determined for the production of each recombinant PRV glycoprotein. Confirmation of glycosylation of recombinant gB, gC and gD suggested their usefulness as antigens for the development of diagnosis kit or vaccines for Aujeszky's disease.

• IMMUNE NETWORK
• /
• v.4 no.1
• /
• pp.7-15
• /
• 2004

Background: The heat shock proteins (HSPs) play an important role in cellular protection mechanisms against physical or chemical stresses. In this study scFv antibodies specific for human HSP70.1 were isolated from a semi-synthetic human scFv library with the ultimate goal of developing anti-HSP70.1 intracellular antibody (intrabody) that may offer an attractive alternative to gene targeting to study the function of the protein in cells. Methods: A semi-synthetic human scFv display library ($5{\times}10^{8}$ size) was constructed using pCANTAB-5E vector and the selection of the library against bacterially expressed recombinant human HSP70.1 was attempted by panning. Results: Three positive clones specific for recombinant HSP70.1 were identified. All three clones used $V_{H}$ subgroup III. On the other hand, $V_{L}$ of two clones belonged to the kappa light chain subgroup I, but the other utilized $V_{k}$ subgroup IV Interestingly, these scFv molecules specifically reacted to the recombinant HSP70.1, yet failed to recognize native HSP70 induced in U937 human monocytic cells by heat treatment. Conclusion: Our results indicated that affinity selection of an scFv phage display library using recombinant antigens produced in E. coli might not guarantee the isolation of scFv antibody molecules specific for a native form of the antigen. Therefore, the source of target antigens needs to be chosen carefully in order to isolate biofunctional antibody molecules.

• International Journal of Oral Biology
• /
• v.43 no.4
• /
• pp.201-207
• /
• 2018

Porphyromonas gingivalis is among the major etiological pathogens of chronic periodontitis. The virulence mechanisms of P. gingivalis is yet to be identified as its activity is largely unknown in actual disease process. The purpose of this study is to identify antigens of P. gingivalis expressed only in patients with chronic periodontitis using a unique immunoscreening technique. Change Mediated Antigen Technology (CMAT), an antibody-based screening technique, was used to identify virulence-associated proteins of P. gingivalis that are expressed only during infection stage in patients having chronic periodontitis. Out of 13,000 recombinant clones screened, 22 tested positive for reproducible reactivity with rabbit hyperimmune anti-sera prepared against dental plaque samples acquired from periodontitis patients. The DNA sequences of these 18 genes were determined. CMAT-identified protein antigens of P. gingivalis included proteins involved in energy metabolism and biosynthesis, heme and iron binding, drug resistance, specific enzyme activities, and unknown functions. Further analysis of these genes could result in a novel insight into the virulence mechanisms of P. gingivalis.

• Parasites, Hosts and Diseases
• /
• v.42 no.4
• /
• pp.195-200
• /
• 2004

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.

• IMMUNE NETWORK
• /
• v.7 no.1
• /
• pp.26-30
• /
• 2007

Background: Mycobacterial antigens released as PIM, LM, LAM, lipoproteins and other cellular factors may contribute to macrophage and dendritic cell activation through pattern recognition receptors such as TLRs. In this study, we assessed cytokine production and ERK activation with stimulation of several major mycobacterial antigens. Methods: Purified mycobacterial antigens (10, 22, 30, 38kDa) and recombinant antigens (6, 16, 19, 38kDa, Ag85A antigen) were studied. The production of cytokines (TNF-${\alpha}$, IL-12, IL-6) was measured by ELISA. The ERK activation was detected by western blotting. The expression of TLR2 or TLR4 was measured by flow cytometry. Results: Among purified antigens only 30kDa antigen induced production of IL-6 or TNF-${\alpha}$ in THP-1 macrophage cells. When THP-1 macrophage cells were treated with 30kDa antigen, phosphorylation of ERK was detected. ERK activation also occurred in TLR2 transfectant HEK293 cells with 30kDa antigen stimulation. Conclusion: 30kDa antigen is one of the major mycobacterial antigens inducing cytokine production and MAP kinases phosphorylation in macrophages.

• Korean Journal of Veterinary Research
• /
• v.29 no.3
• /
• pp.407-416
• /
• 1989

The prospect of live vaccines consisting of genetically modified vaccinia virus expressing foreign genes is exciting, but important issues concerning safety and efficacy need to resolved. Vaccinia virus (VV) is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. The WHO smallpox eradication program, involving the extensive use of VV vaccines, resulted in the late 1970s in the elimination of one of the world's most feared diseases. This achievement is a triumph for preventive medicine and for international collaboration in public health. In 1980, WHO recommended that the routine use of smallpox vaccine should be stopped. Against this background, the prospect of li ve vaccines consisting of genetically modified VV expressing foreign antigens arising from the work of Moss, and Paoletti and their colleagues in 1982 has been greeted with enthusiasm. These investigators have shown that genes coding for immunogenic proteins can be inserted into VV DNA without impairing the ability of the virus to grow in cell culture. Moreover experimental animals infected with VV recombinants containing genes coding for a variety of immunizing proteins have been shown to be protected against challenge infection with the corresponding infectious agent. In this communication, I describe current progress in the construction of a novel plasmid vector that facilitate the insertion and expression of foreign genes in VV as well as the selection of recombinants.

• BMB Reports
• /
• v.49 no.6
• /
• pp.331-336
• /
• 2016

In murine collagen-induced arthritis (CIA), self-reactive T cells can recognize peptide antigens derived from type-II collagen (CII). Activation of T cells is an important mediator of autoimmune diseases. Thus, T cells have become a focal point of study to treat autoimmune diseases. In this study, we evaluated the efficacy of recombinant MHC class II molecules in the regulation of antigen-specific T cells by using a self peptide derived from CII (CII260-274; IAGFKGEQGPKGEPG) linked to mouseI-A^q in a murine CIA model. We found that recombinant I-A^q/CII260-274 molecules could be recognized by CII-specific T cells and inhibit the same T cells in vitro. Furthermore, the development of CIA in mice was successfully prevented by in vivo injection of recombinant I-A^q/CII260-274 molecules. Thus, treatment with recombinant soluble MHC class II molecules in complex with an immunodominant self-peptide might offer a potential therapeutic for chronic inflammation in autoimmune disease such as rheumatoid arthritis.

• Parasites, Hosts and Diseases
• /
• v.54 no.2
• /
• pp.239-241
• /
• 2016

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.5
• /
• pp.621-629
• /
• 2022

Bacterial outer membrane vesicles (OMVs) typically contain multiple immunogenic molecules that include antigenic proteins, making them good candidates for vaccine development. In animal models, vaccination with OMVs has been shown to confer protective immune responses against many bacterial diseases. It is possible to genetically introduce heterologous protein antigens to the bacterial host that can then be produced and relocated to reside within the OMVs by means of the host secretion mechanisms. Accordingly, in this study we sought to develop a novel platform for recombinant OMV (rOMV) production in the widely used bacterial expression host species, Escherichia coli. Three different lipoprotein signal peptides including their Lol signals and tether sequences-from Neisseria meningitidis fHbp, Leptospira interrogans LipL32, and Campylobactor jejuni JlpA-were combined upstream to the GFPmut2 model protein, resulting in three recombinant plasmids. Pilot expression studies showed that the fusion between fHbp and GFPmut2 was the only promising construct; therefore, we used this construct for large-scale expression. After inducing recombinant protein expression, the nanovesicles were harvested from cell-free culture media by ultrafiltration and ultracentrifugation. Transmission electron microscopy demonstrated that the obtained rOMVs were closed, circular single-membrane particles, 20-200 nm in size. Western blotting confirmed the presence of GFPmut2 in the isolated vesicles. Collectively, although this is a non-optimized, proof-of-concept study, it demonstrates the feasibility of this platform in directing target proteins into the vesicles for OMV-based vaccine development.

• Parasites, Hosts and Diseases
• /
• v.51 no.6
• /
• pp.751-754
• /
• 2013

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.