• 제목/요약/키워드: Antibody identification test

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동남참게(Eriocheir japonicus)의 난황단백질 정제와 그 전구체의 합성부위 구명 (Purification of the Yolk Protein, and Identification of the Synthetic Site of Its Precursor in Eriocheir japonicus (Decapoda, Brachiura))

  • 한창희;배현환
    • 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.

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Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting

  • Yang, Zhen-quan;Xue, Yu;Rao, Sheng-qi;Zhang, Mi;Gao, Lu;Yin, Yong-qi;Chen, Da-wei;Zhou, Xiao-hui;Jiao, Xin-an
    • Journal of Microbiology and Biotechnology
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    • 제27권11호
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    • pp.1971-1982
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    • 2017
  • Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

서울지역에서 문제되고 있는 강아지의 급성설사증에 관한 병인학적 연구 (Etiologic Survey on the Acute Diarrhea of Puppies in Seoul)

  • 한홍율;박희명;이진희;오태호;정순욱;윤신근;박철만
    • 한국임상수의학회지
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    • 제10권2호
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    • pp.171-184
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    • 1993
  • In Seoul area, there are so many kennel clubs, veterinary hospital, pet establishment and breeding confers that various problems have occurred. They are crowed pet houses, poor sanitation, stress to puppies, sudden environmental changes to puppies and unvaccination against parvovirus, canine distemper virus, parainfluenza virus, infectious hepatitis caused by Adenovirus type I and Leptospira. Several studies were made to survey the infectious agents involved in acute diarrhea of poppies in Seoul are, such as history taking, physical examination, complete blood count and serum chemistry, histopathological finding, bacterial isolation and identification, and hemagglutination test in feces and hemagglutination inhibition test in serum against parvovirus, respectively. The results obtained are summarized as followed. 1. The percent of PCV (30.5$\pm$5.6) and concentration of Total protein(5.0$\pm$0.8) resulted from statistical analysis are significantly lower than normal values (p<0.05), respectively. In addition, fibrinogen (505$\pm$326) was significantly higher an normal value (p<0.001), Band neutrophil (22.9$\pm$12.7) showed signifiant difference (P<0.01). decreased monocyte (3.2$\pm$2.1) and eosinophil (0.7$\pm$0.8) values appeared statistically significant (p<0.001), lymphocyte (16.7$\pm$9), as well. 2. The concentrations of calcium .(8.0$\pm$2.8), glucose (40.1$\pm$31.4), and albumin (2.0$\pm$0.39) were lower than normal values (p<0.01, p<0.01, p<0.001), respectively. Also Inorganic phosphate (7.1$\pm$2.4), pH (p<7.9f:0.2), and Blood Urea Nitrogen (40.41=37.1) were significantly higher than normal values (p<0.001, p(0.001, p<0.05), respectively. 3. Simple and mixed infections occupied 18% and 82% in the distribution of causes in puppies with acute diarrhea, respectively. 4. As puppies got older, incidence of acute diarrhea caused by Staphylococcus aureus was decreased to 13% and infection of canine distemper virus was increased to 53%, but E coli and canine parvovirus always showed high frequency of outbreak in the body weight ranged from 35g to 7.8Kg. 5. As showed in table 5, infections of E coli and Canine Parvovirus showed high outbreak regardless of the age which is classified into three stages, 35~50 day, 60 day and 75~day to 1 year, canine distemper virus appeared increased, but in case of Staphylococcus aureus, visa versa. 6. In comparison wi methods for the laboratory diagnosis diagnosis parvovirus, Hemagglutination test showed positive reaction in 25% and mean serum antibody titer measured by Hemagglutination inhibition test showed 2779 (n=20). In addition, positive reaction was 90% (18/20). 7. In histopathological studies, enteritic and pneumonic lesions were indicated in 53.7% and 39.5% of samples, respectively. 8. Etiologic diagnosis based on the history taking, clinical signs and histopathological findings in puppies with acute diarrhea and vomiting indicated that 50% of puppies were infected by canine parvovirus and distemper virus. 9. In the parasitological examination made by simple flotation with saturated zinc sulfate tour parasites found were Isospora canis, Toxocara canis, Ancylostoma spp and Toxocara spp. Isospora canis and Toxocara canis were more frequently found among those parasites of diarrhoeic causes in puppies ranged from 35 days to 1 year. Their infestation rates were 15% and 13% respectively.

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돼지 유래의 β-1,4-N-acetylglucosaminyltransferase A (MGAT4A) 유전자의 동정 및 기능 분석 (Identification and Functional Analysis of Pig β-1,4-N-Acetylglucosaminyltransferase A (MGAT4A))

  • 김지윤;황환진;정학재;박미령;변승준;김경운
    • 생명과학회지
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    • 제26권3호
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    • pp.275-281
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    • 2016
  • 인체치료용 당단백질은 바이오의약품에 있어서 중요한 요소이며 특히, 단백질 말단에 결합되어있는 시알산은 의약품의 체내 활성이나 안정성에 큰 영향을 미친다. 최근 돼지유즙은 바이오의약품을 생산하기 위한 생체반응기로써 주목받고 있다. β-1,4-N-acetylglucosaminyltransferase A (MGAT4A)는 재조합단백질이 가지는 시알산 함량을 늘리기 위한 필수 효소 중 하나이다. 그러나 돼지 MGAT4A는 아직도 그 서열이나 기능이 불분명하다. 따라서 이번 연구에서는 돼지 MGAT4A의 서열 동정 및 기능분석을 하였다. 돼지 MGAT4A는 535개의 아미노산을 코딩하는 1,638개의 염기서열로 이루어져 있으며, 일반적인 당전이효소들의 특징인 type II membrane topology의 특성을 가지고 있다. Real-time PCR분석법을 통하여, 지금까지 알려진 것과는 조금 다르게 MGAT4A 유전자가 간이나 유선에서 많은 발현을 보였으나 소장이나 위, 방광에서는 그 발현량이 적다는 것도 확인하였다. 또한, 효소의 기능 분석을 위하여 PK-15 세포주에서 MGAT4A효소의 과발현을 유도하였다. 과발현이 확인된 세포주로 렉틴을 이용한 면역형광염색법과 ELISA방법으로 MGAT4A효소의 과발현이 β-1,4-N-acetylglucosamine의 함량을 증가시켰음을 확인하였다. 또한 기질반응을 통해 bi-antennary structure에 대한 반응성도 확인하였으며, MGAT4A의 과발현이 유도된 세포에서 약 3배 이상의 높은 기질 반응성을 보였다. 이러한 연구는 생체반응기로써 돼지를 이용하는데 있어서 중요한 기반이 될 것이라고 생각한다.