• 한국동물학회지
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• 제33권1호
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• pp.35-44
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• 1990

생쥐 악하선으로부터 분리한 전 RNA를 poly(U)-sepharose chromatography와 sucrose linear density gradient centrifugation 방법으로 레닌 mRNA를 분리하여 in vitro translation과 immunoprecipitation에 의하여 레닌 mRNA를 확인하였다. 레닌 mRNA로부터 레닌 cDNA를 합성하여 EcoRI inker를 이용하여 pUC19에 삽입시켰고, Taq, polymerase를 이용한 PCR방법으로 합성한 레닌 cDNA는 pUC19의 Hinalll 부의에 삽입하여 재조합 plamid를 각각 작성하였다. 이것을 JM103에 형질전환시켜 레닌 유전자 발현을 유도하여 45,000의 분자량을 갖는 레닌을 얻었으며 이 레닌 단백은 토끼으 혈압을 850115mmHg에서 115-140mmHg로 증가시키는 생리 활성을 나타냈다. 토끼의 신장 DNA를 EMBL3 phage에 삽입시켜 genomic library를 작성한 후, 레닌 cDNA로부터 합성한 probe로 plaque hybridization시켜 genomic 유전자를 갖는 재조합 phage를 분리하였다.

• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.357-363
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• 2013

The identification of bacteriophage proteins on the surface of Lactobacillus rhamnosus Pen was performed by LC-MS/MS analysis. Among the identified proteins, we found a phage-derived major tail protein, two major head proteins, a portal protein, and a host specificity protein. Electron microscopy of a cell surface extract revealed the presence of phage particles in the analyzed samples. The partial sequence of genes encoding the major tail protein for all tested L. rhamnosus strains was determined with specific primers designed in this study. Next, RT-PCR analysis allowed detection of the expression of the major tail protein gene in L. rhamnosus strain Pen at all stages of bacterial growth. The transcription of genes encoding the major tail protein was also proved for other L. rhamnosus strains used in this study. The present work demonstrates the spontanous release of prophage-encoded particles by a commercial probiotic L. rhamnosus strain, which did not significantly affect the bacterial growth of the analyzed strain.

• BMB Reports
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• 제40권2호
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• pp.156-162
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• 2007

Two lysis-defective but DNA synthesis non-defective temperature-sensitive (ts) mutants of mycobacteriophage L1, L1G23ts23 and L1G25ts889 were found to be defective also in phage-specific RNA synthesis in the late period of their growth at 42$^{\circ}C$each to the extent of 50% of that at 32$^{\circ}C$The double mutant, L1G23ts23G25ts889 showed the ts defect in phage RNA synthesis that was nearly additive of those shown individually by the two single-mutant parents. Both G23 and G25 were shown to start functioning sometimes between 30 and 45 min after infection but the former gene might be dispensable after 45 min, while the latter was not. Northern analysis also shows that at 42$^{\circ}C$>, L1G23ts23 affects RNA synthesis more strongly than L1G25ts889 from L1 DNA segments that serve as the template for late gene transcription. Among the 21 virion and 12 non-virion late proteins synthesized by L1, L1G23ts23 is defective in the synthesis of at least 9 virion and all of non-virion proteins at 42$^{\circ}C$>. In contrast, L1G25ts889 is completely defective in synthesis of all the 33 late proteins. Possible roles of G23 and G25 in the positive regulation of transcription of different sets of late genes of L1 have been discussed.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.261-261
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• 1994

Bovine milk xanthine oxidase (E.C.1.1.3.22, XO) purchased from Sigma Chemical Co. had the three protein fragments below 150 kDa on 7.5％ SDS-PAGE, which did not show enzyme activity. To remove these fragments, the enzyme preparation was further purified through Sephadex G-200 column chromatography. Two peaks exhibiting enzymatic activity were separated very closely to the void volume, which were revealed as two different enzyme forms, dimeric and monomeric, confirmed by activity staining on native PAGE. Anti sera-against each of the two enzyme forms were raised by subcutaneous injection at multiple sites on the back of rabbits during 4 weeks. On the immunodiffusion test, it was found that both of the antisera of the two forms could react with each other, which implied that their epitopes were identical In the Western blot analysis of mouse liver cytosol fraction, it was found that rabbit anti-XO antibody bound well with the protein band of monomeric mouse liver XO of about 150kDa. Based on this result, mouse liver cDNA 1 ibrary was screened by in situ hybridizat ion wi th rabbi t anti -XO antibody as probe. Through the immunological screening, recombinant phages giving positive signal by the production of XO were selected and further purified. To validate these clones, purified phages were lysogenized in E. coli Y1089 and their lysates were analysed for enzyme activity and immunoreactivity, It was verified that lysates of the purified recombinant phage lysogens exhibited the enzymatic activity as well as bound wi th XO antibody, when induced by IPTG. The above results assert that selected recombinant phage carries mouse liver XO gene.

• 한국미생물·생명공학회지
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• 제14권2호
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• pp.181-186
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• 1986

대장균 염색체의 acetyl CoA carboxylase (fabE)영역 (염색체 지도상 72분 영역)의 유전자를 가진 결함형질도입 파아지를 분리했다 이 형질도입 파아지로부터 20 Md의 염색체 유래의 DNA를 분리하여 제한효소 지도를 작성했으며 이 영역에는 제한효소 Eco RI의 절단부위는 없었다. 형질도입 파아지 DNA의 제한효소 분해산물들은 pACYC 184 플라스미드 벡터에 재클로닝하여 fab E의 온도감수성 변이를 회복할 수 있는 수 종류의 플라스미드를 분리했다. 이들 플라스미드를 분석하여 fab E 유전자는 7, 4Md Bel II 단편상의 Hind III의 절단부위를 가진 3.4 Md Ban HI-Sal I 단편내에 존재함을 밝혔다.

• Journal of Microbiology
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• 제42권2호
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• pp.126-132
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• 2004

A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30$^{\circ}C$ until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30$^{\circ}C$ for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Topl0F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, com-pared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

• Applied Biological Chemistry
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• 제31권3호
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• pp.219-225
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• 1988

Avian Sarcoma Virus의 plasmid DNA중(中)의 역 이사효소의 유전자(遺傳子)를 온도의존성(溫度依存性) 발현(發現) vector인 pPL-lambda에 cloning하여 온도(溫度)에 민감한 phage ${\lambda}$의 repressor인 cI857 gene을 갖고 있는 bacteriophage lysogen인 N4830에 transformation시켰다. transformant를 pL promoter의 발현(發現)을 억제(抑制)하는 저온(低溫)$(28^{\circ}C)$에서 배양(培養)시킨 뒤, 이 repressor를 억제(抑制)하여 transcription을 촉진(促進)하게 하는 고온(高溫)$(42^{\circ}C)$에서 배양(培養)시킨 다음 균체(菌體)를 회수(回收)하여 RNA를 추출(抽出)하고 분석(分析)을 한 결과(結果) 도입(導入)된 역전사 효소 유전자(遺傳子)의 전사(轉寫)가 고온(高溫)에서 증대(增大)되었다.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1996년도 한국생물과학협회 국제학술대회
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• pp.195.2-195
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• 1996

acs gene cloning was constructed by subcloning the 2.2-kb MunI-MunI restriction fragment of 638 and 639 which include acs gene from the kohara phage into the unique EcoRI site of pUC18 and pJM9131 containing the PHA biosynthesis genes. Then recombinant E. coli fadRatoC(Con) mutants containing the polyhydroxyalkanoate(PHA) biosynthesis genes are able to incoporate s significant levels of 3-hydroxyvalerate (3HV) into the copolymer [P(3HB-co-3HV)]. Quantitative determination of PHB and P(3HB-co-3HV) was performed by gas-chromatographic analysis of extracts obtained from methanolysis of lyophilized cells.

• The Korean Journal of Physiology and Pharmacology
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• 제1권2호
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• pp.195-200
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• 1997

Water transport in highly-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP). AQP1 (AQP-CHIP) is the first recognized aquaporin identified from red cells and renal proximal tubules. Up until now 4 other aquaporin homologs have been reported. Each aquaporin has its unique tissue distribution and regulatory mechanims. To elucidate molecular mechanisms for their transcription regulation and tissue-specific expression isolation of aquaporin genes is required. To clone promoters of the AQP family mouse genomic library was screened by the 1st exon-specific probe of AQP4, and 5 different plaques were positively hybridized. Phage DNAs were purified and characterized by restriction mapping and sequencing. One of them is the mouse AQP-CD gene. The gene was consisted of 4 exons and the exon-intron boundaries of mouse AQP-CD gene were identified at identical positions in other related genes. The 5'-flanking region of AQP-CD gene contains one classic TATA box, a GATA consensus sequence, an E-box and a cyclic AMP-responsive element. The cloning of the mouse AQP-CD gene, of which product is expressed in the collecting duct and is responsible for antidiuresis by vasopressin, will contribute to understand the molecular mechanisms of tissue-specific expression and regulation of AQP-CD gene under various conditions.

• 대한수의학회지
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• 제39권4호
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• pp.797-805
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• 1999

T sergenti cDNA library were constructed to get a more broad information about the structural, functional or antigenic properties of the proteins, and analyzes for their partial cDNA sequences and expression sequences tags(ESTg). The mRNA were purified from T sergenti isolates to identify the information of antigen gene, then first and second strand cDNA was synthesized. EcoR I adaptor ligation and Xho I enzyme restriction were used to the synthesized cDNA, and ligated into a Uni-ZAP XR vector. T sergenti cDNA library was constructed with packaging and amplification in vitro. Antibody screening was performed with constructed T sergenti cDNA library using antisera against T sergenti. Among those clones, eight phagemids were rescued from the recombinant in vivo excision with f1 helper phage. Using the analysis of endonuclease restriction and PCR, the recombinant cDNA were proved having a 0.5-3.0kb of inserts. The eight of partial cDNA clones' sequences were obtained and examined for their homology using BLASTN and BLASTX. The eight of sequenced clones were classified into three groups according to the basis of database searches. A total 3,045bp of partial cDNA sequence were determined from six clones. The putatively identified clones contain a cytochrome c gene, a heat shock protein gene, a cyclophilin gene, and a ribosomal protein gene. The unidentified clones have a homology to ATP-binding protein(mtrA) gene of S argillaceus, DNA-binding protein(DBP) gene of Pseudorabies virus 85kDa merozoite protein gene of B bovis, mRNA spm1 protein of T annulata and glycine-rich RNA-binding protein mRNA of O sativa etc.