• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.194-199
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• 2002

Many oxidative metabolites of tetrahydrocannabinols (THCs), active components of marijuana, were pharmacologically active, and 11-hydroxy-THCs, 11-oxo-${\Delta}^8$-THC, 7-oxo-${\Delta}^8$-THC, 8$\beta$, 9$\beta$-epoxyhexahydrocannabinol (EHHC), 9$\alpha$, l0$\alpha$-EHHC and 3'-hydroxy-${\Delta}^9$-THC were more active than THC in pharmacological effects such as catalepsy, hypothermia and barbiturate synergism in mice. Cannabidiol (CBD), another major component, was biotransfomred to two novel metabolites, 6-hydroxymethyl-${\Delta}^9$-THC and 3-pentyl-6, 7, 7a, 8, 9, lla-hexahydro-I, 7-dihydroxy-7, 1O-dimethyldibenzo[b, d]oxepin (PHDO) through 8R, 9-epoxy-CBD and 85, 9-epoxy-CBD, respectively. Both metabolites exhibited some pharmacological effects comparable to d9 - THe. Cannabinol (CBN), the other major component, was mainly metabolized to ll-hydroxy-CBN by hepatic microsomes of animals including humans. The pharmacological effects of the metabolite were higher than those of CBN demonstrating that II-hydroxylation of CBN is metabolic activation pathway of the cannabinoid as is the case in THCs. Tolerance and reciprocal cross-tolerance developed to pharmacological effects d8 - THC and ll-hydroxy-d8-THC , and the magnitude of tolerance development produced by the metabolite was significantly higher than that by d8-THC. The results indicate that ll-hydroxy-d8-THC has an important role not only in the pharmacological effects but also its tolerance development of d8 - THe. THCs and their metabolites competed to the specific binding of CP-55, 940, an agonist of cannabinoid receptor, to synaptic membrane from bovine cerebral cortex. The Ki value of THCs and their metabolites were closely paralleled to their pharmacological effects in mice. A novel cytochrome P450 (cyp2c29) was purified and identified as a major enzyme responsible for the metabolic activation of d8-THC at the II-position in the mouse liver. cDNA of CYP2C29 was cloned from a mouse cDNA library and its sequence was determined. The oxidation mechanism of THC by cyp2c29 was proposed.

• IMMUNE NETWORK
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• 제2권4호
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• pp.233-241
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• 2002

Background: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. Methods: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain ${\lambda}$ genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. Results: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. Conclusion: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.

• 농업과학연구
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• 제45권2호
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• pp.248-257
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• 2018

Overexpression of AtCYP78A7, a gene encoding a cytochrome P450 protein, has been reported to improve tolerance to drought stress in genetically modified (GM) rice (Oryza sativa L.). The aim of this study was to evaluate the potential allergenicity and acute oral toxicity of the AtCYP78A7 protein expressed in GM rice. Bioinformatics analysis of the amino acid sequence of AtCYP78A7 did not identify any similarities with any known allergens or toxins. It showed that no known allergen had more than a 35% amino acid sequence homology with the AtCYP78A7 protein over an 80 amino acid window or more than 8 consecutive identical amino acids. The gene encoding the AtCYP78A7 protein was cloned in the pGEX-4T-1 vector and expressed in E. coli. Then, the AtCYP78A7 protein was purified and analyzed for acute oral toxicity. The AtCYP78A7 protein was fed at a dose of 2,000 mg/kg body weight in mice, and the changes in mortalities, clinical findings, and body weight were monitored for 14 days after the dosing. Necropsy was carried out on day 14. The protein did not cause any adverse effects when it was orally administered to mice at 2000 mg/kg body weight. These results indicate that the AtCYP78A7 protein expressed in GM rice would not be a potential allergen or toxin.

• The Plant Pathology Journal
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• 제25권3호
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• pp.225-230
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• 2009

Lily mottle virus (LMoV) causes flower quality reduction in Lilium spp. The coat protein gene was RT-PCR-amplified from total RNA extracted from infected lily leaves and the amplified fragment was cloned into the pRSET expression vector tagged with a His-MBP. The plasmid of recombinant coat protein was used to transform an Escherichia coli strain pLysS and was expressed. The coat protein was purified by affinity chromatography using a Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE. The in vitro-expressed protein was used for immunization of mice. The polyclonal and monoclonal antibodies reacted specifically for the detection of LMoV in lily extracts in Western blot. Moreover the monoclonal antibodies reacted with lily extracts in DAS-ELISA with no unspecific or heterologous reactions against other non-serologically related viruses, but the polyclonal antibodies revealed a weak reaction against both infected lily and healthy control.

• BMB Reports
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• 제34권2호
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• pp.156-165
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• 2001

Leptin is a circulating non-glycosylated protein that is mainly produced in adipocytes. Leptin acts in the brain to regulate food intake and energy expenditure. Previously we reported our success in the isolation of a partial cDNA of the long form of the leptin receptor, OB-Rb, from rat spleen, and showed that leptin might also play a role in peripheral immune organs. In the present study, for the first time, the complete coding region of OB-Rb cDNA was cloned from rat splenocytes, and its nucleotide sequence was determined. The cDNA was then further expressed in E. coli and mammalian cells, thereby confirming the functional integrity of this receptor. Prokaryotically overexpressed OB-R protein was then used as an immunizing antigen in BALE/c mice to produce leptin receptor-specific antibodies. By using them, we confirmed the cell surface expression of OB-Rb in transfected CHO cells. It is our belief that the reagents, as produced in this study, will be of great use in further studies of the biological role of rat leptin.

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.494-498
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• 1989

AIDS 환자의 치명적인 2차 감염을 유발하는 Cryptosporidium parvum 의 Infective stage 인 sporozoites의 단일군 항체를 분리하였다. Oocysts를 효소처리하여 sporozoites를 excystation시킨 후 Isopycnic percoll gradients를 이용하여 sporozoites를 순수분리한 후 단일군 항체 생산을 위한 항원으로 사용하였다. 두 달된 BALB/c 쥐를 immunize한 후 splenocytes와 P3-X63-Ag8 myeloma cells를 융합시킨 후 hybridoma 기술을 이용해 Kor1(IgGl), Ea2(Ig2a) 두 clones을 분리하였으며 정제된 sporozoites를 SDS-PAGE로 분리한 후 Western blot을 이용하여 단일군 항체 Kor1과 Ea2는 20,000 daltons 크기의 항원을 인식하였다. Immunofluorescent assay에서 단일군 항체가 sporozoites 표면에 반응하는 것으로 보아 20-kDa 단백질 항원은 sporozoites 표면에 위치하는 항원으로 밝혀졌으며 C. parvum에 감염되었을 때 항체생성에 관여하는 중요 항원 중 하나일 것으로 추정되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제27권9호
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• pp.1355-1359
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• 2014

To detect goat vascular endothelial growth factor (VEGF)-mediated regrowth of hair, full-length VEGF164 cDNA was cloned from Inner Mongolia cashmere goat (Capra hircus) into the pET-his prokaryotic expression vector, and the recombinant plasmid was transferred into E. coli BL21 cells. The expression of recombinant $6{\times}his-gVEGF164$ protein was induced by 0.5 mM isopropyl thio-${\beta}$-D-galactoside at $32^{\circ}C$. Recombinant goat VEGF164 (rgVEGF164) was purified and identified by western blot using monoclonal anti-his and anti-VEGF antibodies. The rgVEGF164 was smeared onto the dorsal area of a shaved mouse, and we noted that hair regrowth in this area was faster than in the control group. Thus, rgVEGF164 increases hair growth in mice.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.1034-1040
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• 2009

Porin proteins of Gram-negative bacteria are outer membrane proteins that act as receptors for bacteriophages and are involved in a variety of functions like solute transport, pathogenesis, and immunity. Salmonella enterica serovar Typhi (S. Typhi), a Gram-negative bacterium, is the causative agent of typhoid fever. Porins of S. Typhi have been shown to have a potential role in diagnostics and vaccination. In the present study, the major outer membrane proteins OmpF and OmpC from S. Typhi were cloned in pQE30UA vector and expressed in E. coli. The immunogenic nature of the recombinant porin proteins were evaluated by ELISA by raising hyperimmune sera in Swiss Albino mice with three different adjuvants (i.e., Freund's adjuvant and two human-compatible adjuvants like montanide and aluminium hydroxide gel) and proved to be immunogenic. The recombinant OmpF and OmpC generated in this work may be used for further studies for vaccination and diagnostics.

• 한국미생물·생명공학회지
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• 제41권1호
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• pp.128-134
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• 2013

Salmonella enterica serovar typhi, a Gram-negative food-borne pathogen, causes typhoid fever in humans. OmpC is an outer membrane porin of S. typhi expressed throughout the infection period. OmpC is potentially an attractive antigen for multivalent vaccines and diagnostic kit designs. In this study we combined in silico, in vitro and in vivo approaches to analyze various aspects of OmpC's antigenic properties. The conserved region, in addition to secondary and tertiary structures, and linear B cell epitopes, were predicted. A number of results obtained from in silico analyses were validated by experimental studies. OmpC was amplified, cloned and then expressed, with the recombinant protein then being purified. BALB/c mice were immunized by purified denatured OmpC. The titer of antibody was raised. Results of challenges with the pathogen revealed that the immunity is non-protective. Most of the theoretical and experimental results were in consensus. Introduced linear B cell epitopes can be employed for the design of diagnostic kits based on antigen-antibody interactions.

• 대한수의학회지
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• 제44권4호
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• pp.625-635
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• 2004

Trichnuris suis does not excrete eggs during larval stage as well as in particular adult stage, It is impossible to diagnose by use of fecal examination method in those periods. Therefore, serological diagnostic method can be very useful for those stages. In order to produce monoclonal antibody, specific somatic and secretory-excretory (SE) antigens of T. suis were identified and analyzed by SDS-PAGE and Western blot. Monoclonal antibody-producing hybridoma cells were cloned, which were made of popliteal lymph node of BALB/c mice immunized with a 45 kDa somatic antigen of T. suis. Five clones (1B9, 2C4, n2C5, 2D7 and 2D8) showing strong responses to T. suis antigens were selected and the isotype identified. All monoclonal antibodies were IgG1 isotype and the light chains were k chain. Established monoclonal antibodies reacted specifically to somatic and SE antigens of T. suis and did not cross-reacted to antigens of ascaris suum, trichuris vulpis, or Trichinella spiralis. The sensitivity of somatic and SE antigens against these monoclonal antibodies were significant (p<0.01) associated with those of positive and negative sera.