• 한국가시화정보학회지
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• 제20권3호
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• pp.136-145
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• 2022

Later flow immunoassay (LFIA) is a protein analytical method based on immunoreaction. On the LFIA based protein analytical method, bioreceptor molecule plays a key role, and so a system that evaluates and manages the binding affinity of bioreceptor is needed to secure detection reliability. In this study, Lateral Flow Immunoassay based rapid Bioreceptor Screening Method (rBSM) is presented that provide a simple and quick evaluating method for the binding affinity to the target protein of the antibody as model bioreceptor. To verify this evaluation method, Virus-like particles (VLP) and anti-VLP antibodies are selected as a model norovirus, which is target protein, and the candidate bioreceptors respectively. Among the 5 different candidate antibodies, appropriate antibody could be sorted out within 30 minutes through rBSM. In addition, selected antibodies were applied to two representative LFIA based techniques, sandwich assay and competitive assay. Among these methods, sandwich assay showed more effective VLP detection method. Through applying selected antibodies and techniques to the commercialized mass production lines, an VLP detecting LFIA kit was developed with a detection limit of 10¹² copies/g of VLPs in real samples. Since this proposed method in this study could be easily transformable into other combinations with bioreceptors, it is expected that this technique would be applied to LFIA kit development system and bioreceptor quality management.

• 생명과학회지
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• 제13권4호
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• pp.541-550
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• 2003

본 실험에서는 C형 간염바이러스 (HCV)의 외피 단백질인 E2 당단백질에 결합하는 세포단백질들을 클로닝하기 위해 간세포 cDNA를 phage 표면에 발현시킨 phage library를 제작하였고, 12-mer peptide library와 함께 E2 단백질에 대해 panning을 실시하였다. 검색결과 세포내 신호전달과 cytoskeleton 구성에 관여하는 tensin, membrane protein band 4.1 등 세포질내 단백질과 CCR7, CKR-L2, insulin-like growth factor-1 receptor 등 세포막 단백질 등이 확인되었다. 이들 단백질들을 발현하는 phage들은 수용성 E2단백질을 이용한 결합중화반응 결과 E2 단백질에 특이적으로 결합함이 확인되었다. 사람 T 세포에서 주로 발현되는 CCR7 유전자를 PHA로 활성화된 사람 T 세포의 total RNA를 이용하여 증폭하고 클로닝하였다. 293T 세포에 transfection시켜 단백질 발현양상을 flow cytometer로 분석하여 70% 이상의 세포들이 CCR7을 발현하고 있음을 관찰하였다. 수용성 E2 단백질을 CCR7이 transfection된 세포와 mock transfection된 대조군 세포에 각각 반응시킨 결과 dose-dependent 양상으로 CCR7에 결합하였다.

• 한국산학기술학회논문지
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• 제22권1호
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• pp.431-438
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• 2021

본 연구는 향후 보다 강화될 것으로 예측되어지는 USCG Phase II 형식승인시험을 대비하기 위해 SYBR Green I과 SYBR Gold의 염색 효율을 비교한 후 염색 효율이 높은 시약을 실제 선박평형수처리장치(electrolysis type, UV + electrolysis type)를 통과한 처리수에 적용해서 보았다. 시료의 부피가 0.5 mL ~ 2 mL, 염색 시약(Stock solution)을 100배 및 200배 희석한 조건에서 염색된 바이러스가 가장 선명하게 관찰되었다. SYBR Green I과 SYBR Gold의 염색효율은 장목한 해수조건의 실험구에서 유의한 차이를 보이지 않았지만, SYBR Gold의 노란색에 비해 SYBR Green I으로 염색된 시료에서 발현하는 녹색 형광이 보다 선명해서 관찰이 용이한 것으로 확인되었다. 선박평형수처리장치(electrolysis type, UV + electrolysis type)를 통과하지 않은 실험수 및 대조수에서의 해양 바이러스 현존량은 약 109~1010 VLP 100 mL-1 으로 확인된 반면, 처리수에서는 살아 있는 바이러스가 관찰되지 않았다. 실험수 결과를 보면, SYBR Green I은 해수, 기수, 담수 조건에서 효과적으로 염색이 되는 것으로 확인되었다. 다양한 선박평형수처리기술에 따른 추가적인 검증 및 염색 방법 개발이 필요하지만, SYBR Green I 염색법은 USCG Phase II 미국형식승인시험 바이러스 생산판별에 좋은 대안이 될 수 있을 것으로 판단된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제4권1호
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• pp.66-71
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• 1999

One of the practical limitations with the use of liposomes for delivery of the pharmaceutical substances such antigens is that liposomes are relatively unstable in storage. In order to extend the stability of liposome in storage without affecting their functional activity, solution-type liposomes were dehydrated to form a structurally intact dry liposomes. Comparative immunological evaluation was carried out for both dry and solution-type liposomes containing gag-V3 chimera, consequently it was found that dry liposomes elicited both humoral and cellular response as efficiently as solution-type liposemes did against the same gag-V3 antigen. Especially, long-term stability of the liposomes was remarkably enhanced by the dehydration made to loposomes without a significant change in its ability to elicit immune response in vivo. These results indicate that dry pH-sensitive liposome may become an effective delivery and adjuvant system for general vaccine development.

• 대한수의학회지
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• 제51권2호
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• pp.129-137
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• 2011

Swine diseases could be caused by unrecognized or minor pathogens. In this study, two unknown cytopathogenic agents were isolated from swine, through cell culture. In order to identify these two cytopathogenic agent (designated CP129 and #2045-7), a particle associated nucleic acids PCR (PANPCR) from previous paper was used with simple modification. The cloning procedure was more specified in this study by adding cell control system. According to the modified PAN-PCR, two and four agentsspecific DNA sequences were obtained from CP129 and #2045-7, respectively, and they were identified as Mycoplasma (M.) hyorhinis and Mammalian orthoreovirus by nucleotide BLAST. Since M. hyorhinis (CP129) was filterable and non-visible by microscope, this unusual virus-like nature of M. hyorhinis (CP129) was discussed. Especially, the reovirus (#2045-7) was a serotype 3 and a triple reassortant among three serotypes of reoviruses. It was grouped with recently reported reoviruses from disease cases (swine, human and feline), based on the genetic analysis of L1 and S1 partial sequences. In conclusion, two unknown cytopathogenic agents were successfully identified using modified PAN-PCR with cell control system and they were characterized in this study.

• Journal of Microbiology
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• 제40권4호
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• pp.313-318
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• 2002

To perform the prophylactic study of a vaccine derived from human papillomavirus (HPV) using Balb/c mice, we produced virus like particles consisting of HPV capsid protein L1 which has been reported to induce significant humoral and cellular immunity using various animal model systems. In order to produce HPV16 VLPs, the cDNA of L1 capsid protein in HPV type 16, obtained by polymerase chain reaction, was inserted into yeast expression vector, YEG$\alpha$-HIR525 under the control of GAL10 promoter. The transformation of YEG$\alpha$-HPV16 L1 was performed into the yeast Saccharomyces cerevisiae Y2805 by the lithium acetate method and the yeast clone expressing the highest level of L1 capsid protein of human papillomavirus type 16 was selected by Western blot analysis using anti-HPV16 L1 antibody. The purification of HPV16 VLP has been performed by the ultracentrifugation and gel-filtration methods. To validate the vaccine efficacy of the purified HPV16 VLPs and investigate the properties of HPV16 VLPs to induce humoral immunity, ELISA assay was performed. A significantly increased production of anti-HPV16 VLP antibodies was observed in sera from immunized mice. The neutralization activity of antibodies in the sera from the vaccinated mice was demonstrated by a rapid and simple assay to detect hemagglutihation inhibition activity.

• 한국수산과학회지
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• 제32권1호
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• pp.62-67
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• 1999

1992, 1993년에 경남 통영, 거제 및 전남 여수 등 남해안 주요 굴 채묘 어장에서 참굴, Crassostrea gigas의 극심한 채묘 부진 현상이 발생하여 이에 대한 원인 구명의 일환으로 각 시험 어장 서식 모패 중의 난소기생충 감염율을 조사하고 감염난 및 비감염난에 대한 발생 비교 시험을 실시하였다. 그리고, 모패 중 난의 건강도 판정 및 발생 가능성 추정을 위하여 난 지질 함량을 측정하였다. 또한, 각 채묘 부진 해역에서 채집한 굴 유생 중의 세균의 오염정도와 오염균의 세균상 등을 조사하였다. 1992년 8월에서 1993년 9월 사이 경남 통영 저산, 거제 오수 및 전남 여수 경도, 굴전 지역 굴 암컷 모패의 난소기생충, Marteiliodes chungmuensis 감염 율은 각각 $11.8\~100\%,\;14.3\~100\%,\;$15.4\~93.3\%,\;12.5\~91.7\%$였으나, 충남 대천의 자연산 굴 암컷 모패에서는 난소기생충이 검출되지 않았다. 한편, 난소기생충에 감염된 난의 세포질 내에서는 직경 78$\~$80nm의 바이러스성 입자 (virus-like particle)가 관찰되었다. 난소기생충에 감염된 난을 사용한 인공 발생 실험에서 기생충에 감염된 난은 수정이 불가능하였으며, 동일 생식소에 감염난과 함께 있었던 정상난도 수정은 가능하였으나, $80\%$ 이상이 형태적으로 비정상적인 발생을 하였고, 정상적으로 발생하여 D형 및 초기각정기까지 도달한 유생도 외부 각부터 녹기 시작하여 연체부까지 파괴되는 세포 괴사 현상을 나타내며 전량 폐사하였다. 채묘 부진 현상이 발생한 통영 저산 양식산 참굴의 난지질 함량은 3.8ng/egg이었으나, 충남 대천 자연산의 난지질은 6.2ng/egg였다. 한편, 발생 전 낮은 난 지질 함량을 나타내었던 통영 저산 산의 난은 정상적인 수정, 발생 단계를 거쳐 수정 7일째 초기각정기까지는 발달하였으나, 이후 발생이 진전되지 못한 채 수정 10일째 전량 폐사하였다. 각 시험어장에서 채취한 참굴 유생의 단위개체당 생균수는 거제 오수가 8,100으로 가장 높았고, 통영 저산이 98, 여수 굴전이 5.5로 나타났다. 각 시험어장에서 채수한 해수 중의 생균수는 여수 굴전이 14,000/ml으로 가장 높았으며, 거제 오수와 통영 저산이 각각 370, 260/ml으로 비슷하였다. 채묘 부진 해역에서 채집된 패류 유생 중의 세균상은 Pseudomonas속 및 그 유사세균이 $53.3\~87.1\%$로 절대 우점종을 차지하였다.

• Journal of Pharmaceutical Investigation
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• 제34권3호
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• pp.177-183
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• 2004

Recently, studies on intranasal mucosa delivery of influenza vaccine have been actively developed because of lack of pain and ease of administration. We studied on preparation of nanoparticle delivery system using biodegradable polymer as a poly(DL-lactide-co-glycolide) (PLGA) and their binding characteristics with vaccine. Three kinds of PLGA nanoparticles were prepared by spontaneous emulsification solvent diffusion (SESD) method using sodium dodecyl sulfate and sodium laurate as an anionic surfactant and Lutrol F68 (polyethylene glycol-block-polypropylene glycol copolymer) as a nonionic surfactant. The 5-aminofluorescein labeled vaccine was coated on the surface of nanoparticles by ionic complex. The complexes between vaccine and nanoparticles were confirmed by change of the size. After vaccine coating on the surface of anionic nanoparticles, particle size was increased from 174 to 1,040 nm. However the size of nonionic nanoparticles was not more increased than size of anionic nanoparticles. The amount of coated vaccine on the surface of PLGA nanoparticles was $14.32\;{\mu}g/mg$ with sodium dodecyl sulfate, $12.41\;{\mu}g/mg$ with sodium laurate, and $9.47{\mu}g/mg$ with Lutrol F68, respectively. In conclusion, prepared nanoparticles in this study is possible to use as a virus-like nanoparticles and it could be accept in the field of influenza vaccine delivery system.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.482-488
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• 2019

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. Replicase (Rep) proteins are considered essential for viral replication. Capsid (Cap) protein is the primary immunogenic protein that induces protective immunity. Little is known about comparison on the immunogenicity of PCV2 Rep and Cap fusion protein and Cap protein. In the present study, recombinant baculoviruses expressing the Rep-Cap fusion protein (Bac-Rep-Cap) and the Cap protein (Bac-Cap) of PCV2 were constructed and confirmed with western blot and indirect fluorescence assay. Immunogenicities of the two recombinant proteins were tested in mice. The titers of antibodies were determined with a PCV2-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The $IFN-{\gamma}$ response of immunized mice was measured by ELISA. The mice immunized with the Bac-Rep-Cap and Bac-Cap successfully produced Cap-specific immunoreaction. The mice immunized with the Bac-Cap developed higher PCV2-specific neutralizing antibody titers than mice injected with the Bac-Rep-Cap. $IFN-{\gamma}$ in the Bac-Rep-Cap group was increased compared to those in the Bac-Cap group. Vaccination of mice with the Bac-Rep-Cap showed significantly decreased protective efficacy compared to the Bac-Cap. Our findings will indubitably not only lead to a better understanding of the immunogenicity of PCV2, but also improved vaccines.

• Molecules and Cells
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• 제45권12호
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• pp.911-922
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• 2022

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.