• 대한의생명과학회지
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• 제7권3호
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• pp.103-110
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• 2001

$\alpha$-Fetoprotein(AFP) is a good marker for the detection of several diseases such as hepatocellular carcinoma, gonadal germ cell tumor, gastric tumor, and Down's syndrome. In this study, we developed ELISA, using synthetic peptides corresponding to the epitopes of AFP. Five kinds of peptides were synthesized from AFP to produce antibodies in rats that recognize AFP in human plasma as well as amniotic fluid and do not cross-react with serum albumin. All five kinds of antibodies showed good reactivities with their peptide-keyhole limpet hemocyanin conjugates. Anti-synthetic peptide 1 (R-N-E-Y-G-I-A-S-I-L, 4-13) antibody, in particular, reacted well with AEP as well as synthetic peptide 1-KLH but not with human serum albumin. The binding affinity(Kd) was 2.7$\times$10$^{-9}$M for peptide 1 and 6.8$\times$10$^{-8}$M for AEP. The range for measurement of AFP was 10~1,000 ng/ml. The within-assay and between-assay coefficients of variance(CV) were 4.83% and 10.97%, respectively. In a sample of 31 sera and 33 amniotic fluids, there was a good correlation between AFP values determined in this assay and those in a commercial kit. These results indicate that the antibodies against synthetic peptides corresponding to the epitopes of AFP are highly specific to APP and synthetic peptide-based ELISA would be useful for the measurement of human AFP.

• Parasites, Hosts and Diseases
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• 제51권6호
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• pp.763-766
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• 2013

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.

• Journal of Microbiology and Biotechnology
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• 제29권5호
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• pp.687-695
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• 2019

In a previous work, we performed de novo RNA sequencing of Allomyrina dichotoma using next generation sequencing and identified several antimicrobial peptide candidates based on transcriptome analysis. Among them, a cationic antimicrobial peptide, allomyrinasin, was selected bioinformatically based on its physicochemical properties. Here, we assessed the antimicrobial and anti-inflammatory activities of allomyrinasin against microorganisms and mouse macrophage Raw264.7 cells. Allomyrinasin showed antimicrobial activities against various microbes and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Furthermore, quantitative RT-PCR and ELISA revealed that allomyrinasin reduced cytokine expression levels in the Raw264.7 cells. We also identified inducible nitric oxide synthase, cyclooxygenase-2 expression, and $PGE_2$ production through western blot analysis and ELISA. We confirmed that allomyrinasin bound to bacterial cell membranes via a specific interaction with lipopolysaccharides. Taken together, these data indicate that allomyrinasin has antimicrobial and anti-inflammatory activities as exemplified in lipopolysaccharide-induced Raw264.7 cells. We have provided a potentially useful antimicrobial peptide candidate that has both antimicrobial and anti-inflammatory activities.

• 한국해양바이오학회지
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• 제16권1호
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• pp.55-62
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• 2024

Various antimicrobial peptides (AMPs) from microalgae have shown antibacterial, antiviral, antifungal, anticancer, and antioxidant effects, and play crucial roles in medical applications, aquaculture-related disease management, and the food industry. Magainin 2 (MAG2), an AMP, exhibits high antibacterial and antitumor activity, necessitating an efficient recombinant expression system for low-cost, large-scale production. To enhance MAG2 secretion efficiency in Chlorella, we constructed the SS:MAG2:His vector using the known Chlamydomonas reinhardtii CA1 signal sequence (SS) and obtained a stable transformant via an Agrobacterium-mediated transformation method and RT-qPCR. ELISA results revealed that the MAG2 content secreted into the medium by the SS:MAG2:His transformants increased proportionally with mRNA expression. These findings offer a strategy for high MAG2 secretion in the Chlorella vulgaris platform, potentially minimizing downstream processing costs.

• Journal of Veterinary Science
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• 제23권3호
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• pp.50.1-50.12
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• 2022

Background: There is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity. Objectives: To develop a reliable and rapid strategy to improve diagnostic tools for bTB. Methods: In this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect. Results: The results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity. It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen. Conclusions: Molecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.

• 한국축산식품학회지
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• 제34권3호
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• pp.339-345
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• 2014

This study was performed to develop a rapid immuno-assay kit, by using a specific antigen to detect Hanwoo brand meat. We selected a synthetic antigen specific to our target antibody, named BIO-TAG (Tyr-D-Ala-Phe), by utilizing a computer-based analysis and literature review. BIO-TAG tagged with adjuvant was subcutaneously injected in sheep and Hanwoo. The serum and meat juice of the immunized or non-immunized animal were then analyzed, to measure the titer of antibody by ELISA and Western blot. The amount of antibodies against the BIO-TAG increased (p<0.05) in serum by vaccination. Furthermore, meat juice from the immunized Hanwoo showed greater (p<0.05) antibody titer, compared with those from non-immunized groups. To optimze the dilution factor, we performed dot-ELISA, with various combination levels of BIO-TAG. Results from dot-ELISA showed that 2 mg/mL BIO-TAG was sufficient to distinguish the immunized meat from non-immunized groups. These results support our hypothesis that simple immunization of Hanwoo generates a sufficient amount of antibodies to be detectable in the meat juice by means of the immune-assay. Therefore, specific Hanwoo brand meat can be more precisely identified by our rapid diagnostic kit. This technology can deter possible fraud of counterfeit meat brands in the Korean domestic market with ease and rapidity; and offers a new tool that guarantees consumers high quality Hanwoo brand beef.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.280-287
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• 2015

Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein ($2{\mu}g/mouse$) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility.

• 한국해양바이오학회지
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• 제16권1호
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• pp.45-54
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• 2024

Microalgae have great potential in the biomedical and pharmaceutical industries as a new type of bioreactor that can produce proteins for specific purposes, including recombinant proteins, pharmaceuticals, and industrial enzymes. Despite the production advantages and importance of microalgae-based expression systems, studies on secretion efficiency are limited. In this study, for stable expression and efficient secretion of the heterologous protein (human SCF and human INFγ) in Chlorella vulgaris, we constructed SP:hSCF:His and SP:hINFγ:His plant binary vectors using the signal peptide (SP) of Chlamydomonas reinhardtii, and we obtained stable transformants through the effective agrobacterium-mediated transformation of these vectors. Transformants with accurately inserted hSCF and hINFγ demonstrated stably increased mRNA and protein expression using RT-PCR and western blotting under the same culture conditions. Following the analysis of the proteins secreted into the culture medium using ELISA, it was confirmed that hINFγ was effectively produced in the transformed C. vulgaris culture medium. The overall findings indicate that the combination of heterologous protein and SP may be crucial for ensuring the expression and secretion of recombinant proteins in Chlorella culture systems.

• 생명과학회지
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• 제30권9호
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• pp.804-812
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• 2020

본 연구에서는 아메리카왕거저리에 대한 기능성 연구의 일환으로 아메리카왕거저리 유충의 유전체 분석을 통해 선별된 조포바신 1의 항균 및 항염증 활성을 확인하였다. 선행연구에서 RNA 시퀀싱을 통해 아메리카왕거저리의 전사체를 분석하였으며, 결과를 바탕으로 인실리코(in silico) 분석을 수행하여 전사체 유래 항균 펩타이드를 스크리닝하고 선발하였다. 수행된 항균활성 및 용혈활성 테스트에서 조포바신 1은 세균 및 칸디다 진균에 대해 광범위한 항균활성을 나타낸 반면 마우스 적혈구에 대한 용혈활성은 전혀 없었다. 다음으로 마우스 대식세포주 Raw264.7 세포를 이용하여 조포바신 1의 항염증활성을 확인하였다. 그 결과 조포바신 1은 LPS로부터 유도된 Raw264.7 세포들의 산화질소 생성을 감소시키는 결과를 보여주었다. 뿐만 아니라 실시간 역전사 중합효소 연쇄반응(qRT-PCR) 방법과 효소결합면역흡착측정법(ELISA)을 통해 조포바신 1이 Raw264.7 세포에서 전염증성 사이토카인(IL-6, IL-1β)의 발현을 감소시킨다는 것을 확인할 수 있었다. 또한 염증반응의 신호전달인자들(MAPKs, NF-κB)의 인산화를 억제하는 것을 확인하였다. 게다가 조포바신 1은 LPS와의 상호작용을 통해 결합한다는 것을 확인하였다. 이러한 연구결과들은 아메리카왕거저리 유전체 분석을 통해 확인된 조포바신 1이 항균 및 항염증 치료를 위한 물질로서 개발하는데 가능성이 있을 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.431-437
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• 2016

The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents.