신경세포의 퇴화기전이해와 신경성장인자를 이용한 신경재생촉진 연구(Ⅰ) : Recombinant leukmia inhibitory factor (LIF)-adenoviral vector를 이용한 신경재생 연구

조철만,안상미 국립보건연구원 1999 국립보건원보 Vol.36 No.-

숙주범위가 넓어진 유전자 재조합 핵다각체병 바이러스의 분자생물학적 특성

김우진,우수동,김혜성,진병래,김근영,강석권 한국잠사학회 1996 한국잠사곤충학회지 Vol.38 No.1

To investigate the host range determining factors of nuclear polyhedrosis virus (NPV),Autographa californica NPV and Bombyx mori NPV were coinfected into the two different cell lines, BmN-4 and sf-9. The recombinant baculoviruses, RecS-A6 and RecB-727 which have an expanded host range, were isolated from Sf-9 and BmN-4 cell lines, respectively. The molecular biological characteristics of the recombinant baculoviruses were investigated. The pathogenicity of RecB-727 was similar to that of wild type BmNPV, while the pathogenicity of RecS-A6 was relatively lower than that of wild type BmNPV. The restriction enzyme digestion patterns of parental viruses and recombinant viruses showed that the recombinant virus has an expandedhost range by genetic recombination. Southern blot analysis revealed that the p10 gene of RecB-727 was derived from AcNPV genomic DNA, while RecS-A6 has p10 gene of BmNPV in a viral genome. To investigate the host range expansion mechanism of recombinant baculovirus, HindIII-Sacl 0.6 kb DNA fragments of RecS-A6 and RecB-727 were cloned and sequenced. The results showed that nucleotide sequence in 0.6 kb fragment of recombinant baculoviruses were identical to that of wild type BmNPV helicase gene, suggesting that the expanded host range of recombinant baculoviruses was due to the insertion of BmNPV helicasegene into AcNPV viral genome.

실험실에서 제조한 피부를 이용한 피부자극도의 평가

김방순 ( Bang Soon Kim ),이동윤 ( Dong Youn Lee ),한지현 ( Ji Hyun Han ),이종희 ( Jong Hee Lee ),조광현 ( Kwang Hyun Cho ) 대한피부과학회 2002 大韓皮膚科學會誌 Vol.40 No.12

N/A Background : There is an increasing need for the development of in vitro models capable of substituting for animals in cutaneous irritancy studies, Until now, various culture models have been developed, including skin organ cultures, conventional and air-exposed cell cultures. The air-exposed culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum cerneum layer in these cultures permits the application of potential irritants at the concentrations and formulations which are applied in vivo. Recently, a new human skin recombinant, made of human keratinocytes cultured on de-epidermized dermis with fibroblast-populated collagen matrix, has been developed and appears to represent a better skin equivalent model than previous models. Objective : In the present study, monolayer-cultured human keratinocytes and the new human skin recombinants were evaluated for the test models of various skin irritants. Methods : The extent of skin irritancy induced after application of 3 different irritants (sodium lauryl sulfate, methyl paraben, and polyethylene glycol-400) was evaluated on the basis of (1) MTT assay, (2) neutral red uptake assay, (3) LDH release, and (4) release of IL-1 alpha. In the human skin recombinants, morphological perturbations and changes in the expression of differentiation-specific protein markers (keratin 1, involucrin, filaggrin, and loricrin) were also evaluated. To determine the difference between in vivo and in vitro models for the detection of irritancy, a patch test was performed on 11 normal human volunteers with various concentrations of the different irritants. Results : The results of the present study show that irritant cytotoxicity correlates well with irritant concentration in both monolayer-cultured human keratinocytes and the new human skin recombinant. The new human skin recombinant is superior to monolayer culture as an in vitro model for skin irritancy screening in that the concentrations of test irritants are the same as in vivo. With the human skin recombinant, morphological changes were observed according to the irritant concentration. Conclusion : The new human skin recombinant can be used as an alternative to animals for skin irritancy screening. (Korean J Dermatol 2002;40(12) : 1505∼1517)

Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Outer Membrane Protein L of Salmonella

Fang, Yizhen,Lin, Huixing,Ma, Zhe,Fan, Hongjie The Korean Society for Microbiology and Biotechnol 2016 Journal of microbiology and biotechnology Vol.26 No.7

Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wild-type SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpL-specific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPV-OmpL-vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpL-vaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.

Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Outer Membrane Protein L of Salmonella

( Yizhen Fang ),( Huixing Lin ),( Zhe Ma ),( Hongjie Fan ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.6

Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wildtype SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpLspecific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPVOmpL- vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpLvaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge

Ke Ding,Ke Shang,Zu-Hua Yu,Chuan Yu,Yan-Yan Jia,Lei He,Cheng-Shui Liao,Jing Li,Chun-Jie Zhang,Yin-Ju Li,Ting-Cai Wu,Xiang-chao Cheng 대한수의학회 2018 Journal of Veterinary Science Vol.19 No.2

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccinecan protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase(HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologousrecombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effectof this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutinationinhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation wereincreased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but werenot significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study,a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd(pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

( Shzu Wei Chan ),( Guan Im Ong ),( Sheila Nathan ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.5

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Chan, Shzu-Wei,Ong, Guan-Im,Nathan, Sheila Korean Society for Biochemistry and Molecular Biol 2004 Journal of biochemistry and molecular biology Vol.37 No.5

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

Usefulness of nBos d 4, 5 and nBos d 8 Specific IgE Antibodies in Cow’s Milk Allergic Children

Anna Cingolani,Sabrina Di Pillo,Marzia Cerasa,Daniele Rapino,Nicola Pietro Consilvio,Marina Attanasi,Alessandra Scaparrotta,M. Loredana Marcovecchio,Angelika Mohn,Francesco Chiarelli 대한천식알레르기학회 2014 Allergy, Asthma & Immunology Research Vol.6 No.2

Purpose: The aim of study was to assess the value of recombinants in predicting the degree of symptoms in children with and without anaphylaxisto cow’s milk. Methods: The study included 79 children (70±40 months) referred to the Allergological Unit of the Pediatric Department betweenthe years 2008-2012. Group A was composed of 17 children (78±49.6 months) with anaphylaxis after ingestion of milk. Group B was composed of62 children (73.1±38.6 months) without a history of anaphylaxis, but with less severe symptoms (gastrointestinal and/or skin symptoms). All patientsfrom Group B had a positive open challenge with cow’s milk. All patients underwent an allergic evaluation and blood samples were collectedto test for IgE to recombinans of milk (nBos d 4, 5, 8). Results: A significant difference in nBos d 8 emerged with higher levels in Group A (median[IQR]=2.80 [0.91-16.1]) than B (0.65 [0.24-1.67]; P=0.006), whereas there were no statistically significant differences for nBos d 4 and 5. The recombinants’sum was higher in Group A than B: 8.39 [2.72-41.39] vs 3.04 [1.85-7.31] kUA/L; P=0.044. The recombinant nBos d 8 was superior to the otherrecombinants in identifying children at risk for anaphylaxis, with an area under the curve of 0.718 (95% CI, 0.57-0.86, P=0.006). Considering acutoff of 1.8 kUA/L, nBos d 8 had the most favorable sensitivity and specificity ratio (sensitivity=0.65, specificity=0.77) with an odd ratio of 6.02(95% C.I: 1.89-19.23). Conclusions: This study suggested 2 phenotypes of allergic children, “high-anaphylaxis-risk” and “milder-risk”. These typescan be differentiated through measuring the level of IgE to nBos d 8. Key Words: Child; symptoms; milk; allergy; recombinant proteins

재조합 단백질 생산을 위한 소포체 신호전달

구태원,윤은영,강석우,권기상,권오유,Goo, Tae-Won,Yun, Eun-Young,Kang, Seok-Woo,Kwon, Ki-Sang,Kwon, O-Yu 한국생명과학회 2007 생명과학회지 Vol.17 No.6

ER-Golgi 분비 경로를 통해서 정확한 구조를 가지면서 post-translational modification 과정을 거친 재조합 단백질의 발현을 최대화하는 것은 ER stress반응에 대한 연구의 중요한 계기가 된다. 세포가 스트레스를 받지 않는 상태라도 ER stress signaling은 재조합 단백질의 생산량을 제한하고 품질을 떨어뜨리는 여러 가지 조건을 만들게 된다. ER stress signaling을 막는 여러 가지 방법들이 제시되고 있으며 표 2는 이러한 방법들 중 일부를 나타내고 있다. 일반적으로는 pro-survival 경로에 관련되어 있는 인자를 촉진하고 pro-apoptosis에 관련되어 있는 인자를 억제하는 것들이다. 그러나 ER stress 반응은 매우 복잡하고 적응과 사멸 기작(adaptation and elimination mechanism)의 중간 역할을 하기 때문에 ER stress에 관련된 주요 인자를 산업적으로 응용하기 위해선 이들의 기능에 대해 보다 깊은 연구가 이루어져야 한다. 현재까지 재조합단백질의 생산량을 최대한으로 높이는 방법은 ER stress 반응이 생기지 않도록 fed-batch process를 개선하고 세포 사멸 기작을 조절하며 단백질의 glycosylation 처리를 하는 것이다. The endoplasmic reticulum (ER) is an important intracellular organelle for folding and maturation of newly synthesized transmembrane and secretory proteins. The ER provides stringent quality control systems to ensure that only correctly folded proteins exit the ER and unfolded or misfolded proteins are retained and ultimately degraded. The ER has evolved stress response both signaling pathways the unfolded protein response (UPR) to cope with the accmulation of unfolded or misfolded proteins and ER overload response (EOR). Accumulating evidence suggests that, in addition to responsibility for protein processing, ER is also an important signaling compartment and a sensor of cellular stress. In this respect, production of bio-functional recombinant-proteins requires efficient functioning of the ER secretory pathway in host cells. This review briefly summarizes our understanding of the ER signaling developed in the recent years to help of the secretion capacities of recombinant cells.