• 한국가축번식학회지
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• 제21권2호
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• pp.85-93
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• 1997

These experiments were carried out to examine the development capacity of sexed and then bisected embryo from 8-cell to morula stage. Antisera to histocompatibility-Y(H-Y) antigen were prepared in inbred SD female rat by repeated immunization of spleen cell or testis supernatant from males of same strain. Male and female embryos were separated by delaying development of embryos against H-Y antibody. After sexing, rabbit embryos were bisected and aggregated. The results obtained from the these experiments were summuerized as follows: 1. When mouse and rabbit 8-, 16-cell and morular embryos were culature in H-Y antiserum, the ratio of embryo which has developed to hatching blastocyst was 53.4, 46.3 and 57.4% in mouse embryos, and 49.0, 52.0 and 61.0% in rabbit embryo, respectively. The ratio of mouse and rabbit embryos developed to hatching blastocyst showed nearly natural sex rate(50%), except rabbit mourla showed a little higher ratio(61.0%) as compared to natural sex ratio. 2. When rabbit demi-embryos from 8-, 16-cell embryo and morula were cultured, the percentage of demi-embryos was 70, 68 and 58% without zona pellucida removed, and 62, 69 and 51% with zona pellucida. The rate of aggregation was higher in 8- and 16-cell demi-embryos than in morula demi-embryo. 4. When sexed-demi-embryo was aggregated with another demi-embryo with demi-embryo with same sex, the rate of embryo developed to blastocyst was 60, 50 and 25%, respectively. Eight-cell demi-embryo showed highest rate. In conclusion, it showed that H-Y antiserum which was made by rat spleen cell enabled sexing rabbit embryos. And when rabbit sexed 8-, 16-cell and morula demi-embryo were aggregated, they were developed to eu-blastocyst which suggested the potential of sexed embryo manipulation.

• 한국식품과학회지
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• 제28권3호
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• pp.566-572
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• 1996

본 연구에서는 숙성 중 야기되는 식육의 인화정도를 파악할 수 있는 척도로서 이용하기 위하여, 근원섬유 Z선의 구성단백질의 하나인 zeugmatin에 대한 항체를 조제, 간접연역형광법으로 zeugmatin의 숙성중 변화와 근원섬유의 소편화와의 관계를 알아 보았다. Zeugmatin은 변성하기 쉬운 단백질로서 재래식 방법으로는 정제가 블가능하므로 이 단백질을 함유하는 획분을 정제과정 중에 채취하여 전기영동으로 전개, 이에 해당하는 band를 분리하여 polyclonal항체를 용이하게 받을 수 있었으며, 이 조제된 항체는 zeugmatin과 특이적으로 반응하였다. 조제된 항체를 이용하여 간접면역형광법으로 식육의 숙성중에 zeugmatin의 변화와 근원섬유의 소편화로 나타나는 식육의 연화와의 상관관계를 알아 본 결과, 이 두가지 변화는 특이적으로 일치하였다 즉, 닭의 흉근을 $4^{\circ}C$에서 숙성하면서 측정한 근원섬유의 소편화도는 도살 후 6시간에서 24시간 사이에 급속히 증가하여 이 시간대에 식육이 급속히 부드러워짐을 나타내었고, zeugmatin에 대한 항체가 나타내는 형광광도도 도살 후 6시간에서 24시간 사이의 동일한 시간대에 급적히 약화되어 zeugmatin이 이 시간대에 급속히 인화하였음을 나타내었다. 이상의 결과로 근원섬유 Z선의 구성단백질 중 하나인 zeugmatin은 숙성에 따라 야기되는 식육의 연화정도, 즉 식육의 숙성도를 나타내는 척도로서 이용될수 있으며, 그 이용방법으로는 간접면역형광법이 가장 간편한 방법인 것으로 판단되었다.

• Journal of Microbiology and Biotechnology
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• 제24권5호
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• pp.696-703
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• 2014

Clostridium difficile causes mucosal damage and diarrhea by releasing two exotoxins: toxin A and toxin B. C. difficile colitis is associated with alterations in bowel flora and the failure to mount an effective antibody response. The aim of the current study was to investigate whether antitoxin sera prevent toxin-A-induced apoptosis, cytoskeletal disaggregation, cell detachment, and tight junction loss in cultured colonic epithelial cells. Serum samples were isolated from mice that survived a C. difficile infection following antibiotic treatment, and the antitoxin effects of these samples were investigated in toxin-A-exposed HT29 colonic epithelial cells and a toxin-A-induced animal model of gut inflammation. Unchallenged mice did not produce IgG against toxin A, whereas serum (antiserum) from C. difficile-challenged mice showed significant IgG responses against toxin A. Treatment with the antiserum markedly inhibited mucosal damage and inflammation in the toxin-A-treated mouse model. In contrast to control mouse serum, the antiserum also markedly inhibited toxin-A-induced DNA fragmentation, dephosphorylation of paxillin and Epo receptor (EpoR), deacetylation of tubulin, and upregulation of p21(WAF1/CIP1) and p53. Taken together, these results reveal that the generated antitoxin serum has biotherapeutic effects in preventing various C. difficile toxin-A-induced cellular toxicities.

• 한국가축번식학회지
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• 제12권1호
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• pp.37-41
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• 1988

These studies were carried out to examine the sex of preimplantation mouse embryo. For the investigation of sex-ration of mouse embryos, morula and blastocysts stage embryos treated with H-Y antiserum (10%, v/v) and FITC anti-mouse-IgG were divided into the positive and negative embryos. Positive and negative identified embryos were observed the viability according to the in vitro cultured and the sex ratio was also investigated by chromosomal analysis. The results obtained in these studies were summarized as follows: 1. Two hundred sixty-seven recovered embryos of morula or blastocyst stage were incubated in medium containing H-Y antiserum and FITC anti-mouse-IgG. Positively or negatively identified embryos were 139 and 128. This trend indicated the approximal sex ratio was 1:1. 2. Sex ratio was measured using the embryos treated with indirect immunofluorescence assay to examine the relationship between embryo developmental stage and sex ratio. Sex ratio of morula stage embryos was 45.2% positive and 54.8% negative, on the other hand, the ratio switched to 56.4% positive and 43.6% negative embryo in blastocyst stage. 3. Fourty-seven positive and 57 negative embryos were obtained out of 104 morula stage embryos treated with indirect immunofluorescence assay. Survived positive or negative embryos during in vitro culture were 42 and 49, respectively out of 47 and 57 embryos. 4. The numbers of negative and positive embryos were 171 and 92 out of 163 blastocyst embryos which were incubated in the medium containing H-Y antiserum and FITC anti-mouse-IgG. The result of karyotype test showed the successful rate of sexing embryo is positive and negative embryos was63.0% (58/92) and 62.0% (44/71). The final female to male ratio within 58 positive embryos was 22.7:77.6, and the ratio of the 44 negative embryos was 77.3:22.7.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2135-2145
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• 2015

VP7, an outer capsid protein of grass carp reovirus (GCRV), was expressed and displayed on the surface of Saccharomyces cerevisiae for developing an efficient vaccine against hemorrhagic disease of grass carp. The result of flow cytometry analysis indicated that protein VP7 could be displayed on the surface of yeast cells after inducing with galactose. The expression of VP7 was confirmed by western blot analysis and further visualized with confocal microscopy. The specific antibodies against VP7 generated from mice were detectable from all immune groups except the control group, which was immunized with untransformed yeast cells. The displaying VP7 on glycosylation-deficient strain EBYΔMnn9 was detected to induce a relatively low level of specific antibody amongst the three strains. However, the antiserum of EBYΔM9-VP7 showed relative high capacity to neutralize GCRV. Further neutralization testing assays indicated that the neutralizing ability of antiserum of the EBYΔM9-VP7 group appeared concentration dependent, and could be up to 66.7% when the antiserum was diluted to 1:50. This result indicates that appropriate gene modification of glycosylation in a yeast strain has essential effect on the immunogenicity of a yeast-based vaccine.

• Nutrition Research and Practice
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• 제8권3호
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• pp.278-283
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• 2014

BACKGROUND/OBJECTIVES: Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concerns regarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolytic effects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS: Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimum temperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibition ELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitive patients. RESULTS: Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showed that the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease of more than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could not hydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS: Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However, the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from human patients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis.

• 식물병연구
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• 제12권1호
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• pp.25-27
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• 2006

벼오갈병바이러스(Rice dwarf virus, RDV)는 Phytoreovius 속에 속하며 게놈은 double stranded RNA의 12분절로 이루어져 있는데 전기영동상에서 크기가 큰 것부터 S1에서 S12로 명명한다. 본 실험에서는 RDV 입자 및 P12 단백질의 세포질 내에서의 소재양식을 확인하였다. 바이러스 입자 및 P12 단백질 특이적 항혈청에 protein A-gold 입자를 immunolabelling하여 전자현미경에서 gold입자의 존재양상을 관찰하였다. 바이러스 입자 특이적 항혈청을 사용한 경우에는 gold 입자가 세포질내의 바이러스 입자에 특이적으로 반응한 것이 관찰되었다. P12단백질 특이적 항혈청의 경우 gold 입자가 세포질내의 세포소기관에 특이적으로 존재하지 않고 세포질전체에 산재하여 존재하였다. 이 결과로서 RDV S12가 코드하는 단백질 P12가 세포질내에 존재함을 확인하였다.

• 생명과학회지
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• 제9권3호
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• pp.253-261
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• 1999

Leptin gene, an obesity gene, has been known to involve in the regulation of food intake and body weight. It is also thought to be related to the glucose metabolism, insulin secretion and type II diabetes mellitus. Recently, the production of recombinant leptin protein has been attempted for the application in the treatment of obesity and the correction of hereditary obesity and type II diabetes. In the present study, leptin cDNA was cloned from mouse fat cells by RT-PCR and prokaryotic expression of leptin was attempted in order ot prepare a leptin-specific antigen. Immunization of a rabbit with the leptin-specific antigen into a rabbit resulted in the generation of leptin-specific antiserum that could be useful in the detection of leption expressed in various tissues. The sequence of leptin cDNA prepared in the present study wa identical to the previously reported one. Transformation of E. coli(DH5a) cells with the leptin cDNA-inserted translation vector, pGEX-4T-3-leptin followed by treatment with IPTG (0.1mM) resulted in the expression of a large amount of GST-leptin fusion protein with a molecular weight of 44 KDa as an inclusion body. Denaturation of the insoluble fusion protein by 8M urea, 6M guanidium-HCI or 0.1% 2-mercaptoethanol followed by a slow oxidation could not solubilize the inclusion body. The cell extract was subjected to SDS-PAGE and GST-leptin protein electroeluted from the gel was then injected into a rabbit subcutaneously for the immunization. Anti-GST-leptin rabbit antiserum which had a cross reactivity to the GST-leptin protein was generated. Leptin protein expressed in mouse brain and fat tissues was detected by Western blot immunodetection system using the antiserum generated in the present study.

• Parasites, Hosts and Diseases
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• 제30권2호
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• pp.91-100
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• 1992

폐홉충의 생리식염수 추출액 내에 있는 여러 가지 성분 단백질의 생화학적 성상, 면역학적 특징 그리고 충체 내에서의 생성위치 등의 단세포군 항체나 전기영동을 이용한 연구에서 일부 밝혀졌다. 이 실험은 폐흡충 성충의 생리식염수 추출액 내에 있는 각 성분단백질이 폐흡충의 어느 부위에서 유래한 것인지를 알기 위하여 실시하였다. 먼저 생리식염수 추출액을 8% disc-PAGE로 전기영동하여 성분단백질을 분리하고 각 단백질 대(대)를 포함하는 젤을 잘라 내었다. 이어 전기영동으로 젤에서 성분단백질을 용출(용출)하였다. 각각의 단백질을 토끼에 면역시켜 성분 단백질별 항폐흡충 면역혈청을 만들고 이 항혈청으로 폐흡충 및 감염 고양이 폐의 절편에 면역효소 염색법을 시행하였다. 그 결과 성충추출액으로 면역한 항폐흡충 면역혈청은 폐흡충 성충의 충란, 장관상피세포, 장관 내용물에 강한 반응을 보였고 실질조직에도 염색이 되었다. 그러나 표피, 표피하세포, 고환, 흡판등은 반응이 없었다. 1번 Band 단백질의 항혈청은 충란내 세포에 염색이 되었고 충체에서 배출되어 폐실질 조직에 들어있는 충란에 더욱 강하게 반응하였다. 2번 Band 단백질의 항혈청은 폐홉충의 실질조직에 반응하였다. 3번 Band 단백질의 항혈청은 2번 Band 단백질과 교차반응을 일으켜 염색 반응을 관찰하지 않았다. 4번 Band 단백질의 항혈청은 폐흡충의 장관 내용물과 강한 반응을 보였고 5번 Band 단백질의 항혈청은 장관상피세포에 반응하였다. 6/7번 Band 단백질과 8번 나and 단백질의 항혈청은 각각 실질조직에 반응하였다.

• 한국수산과학회지
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• 제25권5호
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• pp.432-442
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• 1992

동남참게, Eriocheir japonicus의 난황단백전구체 합성부위를 면역조직화학적인 방법으로 구명하기 위하여, 난황단백질을 정제하고, 이 단백질에 대한 항혈청을 제작하여 난황단백질에 대한 면역학적인 성질을 밝힌 후, 난황단백전구체의 항체가 존재하는 항 난황단백혈청을 사용하여 PAP(peroxidase-antiperoxidase)법으로 생식소, 간췌장, 근육조직을 대상으로 면역조직화학 반응을 시켰다. 성숙한 암, 수혈청에 대한 면역전기영동과 Ouch-terlony 면역확산 실험에 의하여 암혈청내에는 암 특이성혈청단백질이 있음이 밝혀졌다. 암, 수혈청에 대한 항혈청과 난황형성중인 암혈청에 대한 조난황단백추출액에 대하여 면역전기영동과 Ouchte-rlony 면역확산 실험을 행한 결과, 암 특이성혈청 단백질은 난황단백전구체임이 밝혀졌다. 조난황단백추출액으로 부터 DEAE 이온교환 chro-matography와 sepharose CL-4B gel 여과법에 의하여 난황단백질을 정제하였으며, 이 난황단백질의 분자량을 sepharose CL-4B gel 여과법에 의하여 추정한 결과 분자량은 약 245,000 dalton이었다. 또한 난황단백질은 면역학적인 분석에 의하여 난황 단백질에 대한 항혈청에는 난황단백전구체에 대한 항체가 존재하고 있음이 증명되었다. 난황단백질에 대한 토끼 항혈청을 이용하여 PAP법에 의한 면역조직화학반응을 시킨 결과, 난황형성중인 난모세포와 성숙한 암참게의 간췌장은 양성반응이 나타는데 반하여 난소내의 여포세포층과 난황형성전 난모세포, 근육층 그리고 성숙한 수참게의 간췌장은 전혀 양성반응이 나타나지 않았다. 따라서 본 종의 난황단백전구체의 합성부위는 간췌장임을 알 수 있다.