• KSBB Journal
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• 제23권4호
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• pp.303-310
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• 2008

소의 혈액, 세포, 조직, 기관 등 소유래 물질을 원료로 사용한 생물의약품, 조직공학제제, 세포치료제의 경우 소유래 원료물질에 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. BPIV3는 동물 세포주, 우혈청 등에 가장 흔하게 오염되는 바이러스이다. 소유래 물질을 원료로 하는 생물의약품, 조직공학제제, 세포치료제 등에서 BPIV3 안전성을 확보하기 위해, 원료물질, 제조공정, 완제품에서 BPIV3를 정량적으로 검출하고, 제조공정에서 BPIV3 제거 검증을 위한 시험법으로 활용이 가능한 BPIV3 real-time RT-PCR 시험법을 확립하였다. BPIV3에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 BPIV3 RNA 정량 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR 민감도는 2.8 $TCID_{50}/mL$이었다. 확립된 시험법의 신뢰성 (reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성 (specificity)과 재현성 (reproducibility)이 우수함을 확인하였다. 확립된 real-time RT-PCR을 생물의 약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 BPIV3를 오염시킨 CHO 세포주와 소유래 콜라겐에서 BPIV3 검출 시험을 실시하였다. BPIV3를 감염시킨 CHO 세포와 세포배양 상청액에서 BPIV3를 정량적으로 검출할 수 있었다. 소유래 콜라겐에서도 7.8 $TCID_{50}/mL$ 까지 정량적으로 검출할 수 있었다. 위와 같은 결과에서 확립된 BPIV3 real-time RT-PCR 시험법은 생물의약품 안전성 보증을 위한 세포주 검증, 생물의약품 생산 공정 검증, 바이러스 제거 공정 검증 등에서 감염역가 시험법과 같은 생물학적 시험법을 대신할 수 있는 신속하고, 특이성과 민감성이 우수한 시험법임을 확인하였다.

• 미생물학회지
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• 제44권3호
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• pp.228-236
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• 2008

세포배양 유래 생물의 약품 생산 공정에서 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. Reovirus type 3 (Reo-3)는 동물 세포주와 동물 세포 배양 공정에 오염되는 대표적인 바이러스이다. 세포배양 유래 생물의약품의 Reo-3 안전성을 확보하기 위해, 세포주, 원료물질, 제조공정, 완제품에서 Reo-3를 정략적으로 검출하고, 제조공정에서 Reo-3 제거 검증을 위한 시험법으로 활용이 가능한 real-time RT-PCR 시험법을 확립하였다. Reo-3에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 Reo-3 RNA 정략 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR 민감도는 $3.2{\times}10^0\;TCID_{50}/ml$이었다. 확립된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성(specificity)과 재현성(reproducibility)이 우수함을 확인하였다. 확립된 real-time RT-PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 Reo-3를 오염시킨 CHO 세포에서 Reo-3 검출 시험을 실시한 결과 Reo-3를 감염시킨 CHO 세포와 세포배양 상청액에서 Reo-3를 정략적으로 검출할 수 있었다. 또한 바이러스필터 공정에서 Reo-3제거 효과를 감염역가 시험법과 비교 검증한 결과 더 빠른 시간에 동일한 결과를 얻을 수 있었다. 위와 같은 결과에서 확립된 Reo-3 real-time RT-PCR 시험법은 생물의약품 안전성 보증을 위한 세포주 검증, 생물의 약품 생산 공정 검증, 바이러스 제거 공정 검증 등에서 감염 역가 시험법을 대신할 수 있는 신속하고, 특이성과 민감성이 우수한 시험법임을 확인하였다.

• 대한소아치과학회지
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• 제32권1호
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• pp.75-88
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• 2005

법랑아세포종은 1868년에 처음 보고된 이래 명칭, 발생기전, 분류 그리고 치료 방법 등에 관하여 수 많은 논란이 있어 왔는데 이는 법랑세포종이 양성종양임에도 불구하고 종양자체의 진행이 파괴적이고, 외과적 처치를 한 후에도 재발이 잘되며, 흔하지는 않지만 악성종양과 유사하게 전이를 보이는 등 독특한 특성을 지니고 있기 때문이다. 정상세포와 암 세포 간에 차이를 보이는 유전자 혹은 정상세포에서 변형이 일어날 때 특이적으로 발현하는 유전의 분리 및 분석하는 것은 암세포의 생성과정을 이해하는데 있어서 중요한 열쇠를 제공할 수 있다. 이에 본 연구는 RNA differential display 방법 중 재연성과 반복성이 개선된 Ordered differential display(ODD)RT-PCR과 보다 개선된 $GeneFishing^{TM}$기술을 이용하여 악성과 양성종양 사이의 유전자 발현의 차이를 조사하고, 특이 유전자의 profile을 확보하고자 하였다. $GeneFishing^{TM}$기술과 RT-PCR을 수행한 결과 nasopharyngeal carcinoma gene을 제외한 9개의 유전자는 악성에서 특이적으로 발현되는 것을 확인하였다. 따라서 $GeneFishing^{TM}$을 이용하면 각 시료간의 mRNA 상에서 발현차이를 보이는 DEG를 비교 분석하면 암관련 유전자, 항생제 태성 유전자, 그리고 분화 관련 유전자들에 대한 연구가 용이하게 수행할 수 있을 것으로 생각된다.

• Journal of Microbiology
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• 제45권2호
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Journal of Microbiology
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• 제44권1호
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• pp.54-63
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• 2006

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the $^{32}P-labeled$ partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5' -untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.

• BMB Reports
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• 제38권6호
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• pp.668-675
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• 2005

A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.2707-2712
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• 2015

Background: The human homologue of the mouse double minute 2 (MDM2) gene is a negative regulator of Tp53. MDM2-309T>G a functional promoter polymorphism was found to be associated with overexpression thereby attenuation of Tp53 stress response and increased cancer susceptibility. We have planned to evaluate the possible role of MDM2-309T>G polymorphism with risk and response to chemotherapy in AML. Materials and Methods: A total of 223 de novo AML cases and 304 age and sex matched healthy controls were genotyped for the MDM2-309T>G polymorphism through the tetra-primer amplification refractory mutation system (ARMS)-PCR method. In order to assess the functional relationship of -309T>G SNP with MDM2 expression level, we quantified MDM2 mRNA in 30 primary AML blood samples through quantitative RT-PCR. Both the (-309T>G) genotypes and the MDM2 expression were correlated with disease free survival (DFS) rates among patients who have achieved complete remission (CR) after first induction chemotherapy. Results: MDM2-309T>G polymorphism was significantly associated with AML development (p<0.0001). The presence of either GG genotype or G allele at MDM2-309 confered 1.79 (95% CI: 1.12-2.86; p<0.001) and 1.46 fold (95%CI: 1.14-1.86; p= 0.003) increased AML risk. Survival analysis revealed that CR+ve cases with GG genotype had significantly increased DFS rates (16months, p=0.05) compared to CR+ve TT (11 months) and TG (9 months) genotype groups. Further, MDM2 expression was also found to be significantly elevated in GG genotype patients (p=0.0039) and among CR+ve cases (p=0.0036). Conclusions: The MDM2-309T>G polymorphism might be involved in AML development and also serve as a good prognostic indicator.

• 미생물학회지
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• 제45권2호
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• pp.105-111
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• 2009

노로바이러스는 급성 위장염을 일으키는 Caliciviridae 과(family)에 속하는 바이러스로 유전자형이 매우 다양하다. 본 연구에서는 노로바이러스 국내분리주의 게놈 RNA로부터 3개의 open reading frame (ORF) 모두의 염기서열을 분석하고, 유전학적 계통분석을 통하여 분자생물학적 특성을 분석하였다. 본 연구에 사용된 노로바이러스(Hu/NLV/Gunpo/2006/KO)는 바이러스성 식중독, 장염 증세를 보이는 2세 여아 가검물로부터 분리되었다. 역전사반응과 PCR 증폭을 통해서 바이러스의 게놈 RNA를 3개의 중첩되는 cDNA 단편으로 합성하였으며, 합성된 cDNA를 염기서열 분석에 직접 사용하였다. 시퀀싱 결과 Hu/NLV/Gunpo/2006/KO는 3개의 ORF (ORF1, 5,100 bp; ORF2, 1,647 bp; ORF3, 765 bp)로 구성되어 있음을 알 수 있었다. 35개의 노로바이러스 국외 분리주와 비교한 결과, ORF1은 ORF2 또는 ORF3에 비해서 상대적으로 염기의 변이율이 낮았으며, 특히 ORF2와 ORF3의 C-말단 부위에서 높은 변이율을 관찰하였다. 유전학적 계통도를 분석한 결과, Hu/NLV/Gunpo/2006/KO는 genogroup II 에 속하며, Saitama U1, Gifu'96, Mc37, Vietnam 026과 같은 클러스터를 형성하는 것을 알 수 있었다. 본 연구를 통하여 노로바이러스 Hu/NLV/Gunpo/2006/KO의 3개의 ORF 염기서열을 모두 밝힘으로써, 앞으로 노로바이러스의 검출법 개발과 유전학적 상관관계뿐 아니라, 유전자의 기능 분석과 관련된 기초연구에 중요한 기초자료를 제공할 수 있을 것으로 기대한다.

• 대한바이러스학회지
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• 제29권1호
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• pp.45-53
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• 1999

We examined the hepatitis G virus infections among 227 Koreans who were healthy or were suspected of hepatitis and determined the phylogenetic relationship based on a part of the NS-5 region of 5 positive samples. Viral RNA was extracted from sera and cDNA was synthesized and subsequently amplified by RT-PCR (reverse transcription-polymerase chain reaction) or RT-nested PCR using random hexamer and NS-5 specific primers (470-20-1-77F, 470-20-1-211R, HGVNESTFO, HGVNESTRE). Five positives were found to belong to samples of patients showing symptoms of viral hepatitis. Primers used for PCR or nested PCR were derived from the NS-5 region. On the other hand, no amplification was detected using primers derived from the 5'-NCR (G-146F, G-401R). We performed TA cloning and sequencing of 5 amplified fragments, and their sequences were compared with those of foreign isolates of HGV. The phylogenetic analysis using MegAlign programme of DNAstar has shown that the Korean isolates are clustered on the phylogenetic tree. In summary, we confirmed the hepatitis G virus infection in 5 cases out of 12 patients showing the symptoms of viral hepatitis. The phylogenetic analysis of sequences of 5 amplified fragments showed that their relations to each other were closer than those to the foreign HGV isolates reported.

• 대한바이러스학회지
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• 제28권3호
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• pp.247-265
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• 1998

To determine the sequence and expression of the VP7 gene of Korean isolates (CAU-9), viral RNA was purified and used for cDNA amplification by RT-PCR. The VP7 cDNA was cloned, sequenced, and expressed using baculovirus expression system. The result showed that the sequence homologies CAU-9 compared with foreign isolated strains Wa, 417, TMC-II, 95B and SA11 were ranged from 74.0% to 95.1 % of nucleotide sequence and 35% to 43% of amino acid sequence, respectively. High homology of CAU-9 was observed in Japanease isolates 417 (nucleotide sequence homology was 95.1% and amino acid sequence homology was 43%). To express VP7 gene, the VP7 cDNA was cloned into pCR-Bac vector and inserted into the genome of baculovirus adjacent to the polyhedrin promoter by cotransfection of Spodoptera frugiperda (Sf9) insect cells with wild type baculovirus DNA. In antigenic analysis of Sf9 cells inoculated with the recombinant VP7, immunofluorescence assay revealed positive for viral antigens. In metabolic labeling of Sf9 cell lysates infected with recombinant baculoviruses, it was revealed that the protein of 34 kDa was expressed. The limited study of expressed VP7 protein inoculated with guinea pigs failed to elicit neutalizing antibody. As a results, the sequence analysis and expression of VP7 protein of rotavirus CAU-9 isolated from an infant in Korea could permit the conformation and development of virus like particles which may be useful in designing vaccine strategy.