• Archives of Pharmacal Research
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• 제27권7호
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• pp.776-780
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• 2004

Interferons are cytokines that confer resistance to viral infection and inhibit cellular proliferation. The interferon alpha gene from human blood samples was amplified, cloned and expressed in E. coli (BL21). Leukocyte chromosomal DNA was used as a source of template DNA. Using specific primers, the gene for HulFN$\{alpha}$-2b was amplified and inserted into the E. coli vector, pET21b, by ligation of the HindIII and BamHI linkers of the vector and insert. The insert was further analyzed by PCR, DNA restriction mapping and sequencing, and expressed in a suitable E. coli strain. The production of this important cellular protein in the laboratory has significant applications in production of the recombinant pharmaceutical proteins.

• 대한수의학회지
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• 제60권3호
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• pp.109-116
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• 2020

This study aimed to develop a semi-nested polymerase chain reaction assay for the direct detection of Mycoplasma synoviae (M. synoviae) from clinical samples using three newly designed oligonucleotide primers specific to the variable lipoprotein haemagglutinin (vlhA) gene and differentiate M. synoviae field strains based on a nucleotide deletion or the insertion of the proline-rich repeat (PRR) region of the vlhA gene. The developed semi-nested polymerase chain reaction (PCR) assay revealed positive results in 12 out of 100 clinical samples collected from chickens showing lameness and joint swelling. Six positive samples were selected randomly for sequencing, and sequence analysis revealed 96.3-100% nucleotide identities compared to the reference sequences. Phylogenetic analysis showed that sequences of the strains in this study were closely related to WVU1853 (Spain), CK.MS.UDL.PK.2014.2 (Pakistan), and F10-2AS (USA) strains, but they were distinct from the M. synoviae-H vaccine strain sequence. M. synoviae obtained from these samples were identified as types A and C with a length of 38 and 32 amino acids, respectively. These results indicated that the specific and sensitive semi-nested PCR could be a useful diagnostic tool for the direct identification of clinical samples, and the sequence analysis of the partial vlhA gene can be useful for typing M. Synoviae.

• Plant Resources
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• 제4권1호
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• pp.41-47
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• 2001

This study was conducted to investigate the occurrence of Soil borne wheat mosaic virus(SbWMV) in barley fields in Korea and to examine the host pathogenicity of SbWMV. By using the ELISA test, SbWMV was detected in the six regions : Suwon, Milyang, Jinju, Youngkwang, Iksan, and Chonju. SbWMV was isolated from the two strains, Albori strain from Jinju and Eunpamil strain from Milyang. SbWMV was collected from leaves showing mosaic, yellowing and necrosis stripes. SbWMV was inoculated mechanically on 1∼1.5 leaf stages with leaf-rubbing to identify the host pathogenicity of 36 Korean barley cultivars, a wheat cultivar, two rye cultivars, three Japanese barley cultivars and Chenopodium amaranticola. Viral sympoms of inoculated leaves appeared on moulted loaves about 4 to 6 weeks of inoculation. Baegdong and Tapgolbori, infected from Albori strain and Eunpamil strain infected from Samdobori showed much higher susceptibility than C. amaranticola and C. quinoa which showed ring spots and chlorotic spots respectively. Virus particles were observed by the electron microscope. They were rod-shapes, which are bipartite, of 142 nm or 281 nm in length with 20 nm diameter on infected leaves. Specific detection and identification of SbWMV was set up using the RT-PCR. PCR fragments of SbWMV(0.5kb) were obtained by using the designed primers for SbWMV RNA 2.

• 생명과학회지
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• 제30권1호
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• pp.1-9
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• 2020

키위 세균성 궤양병의 원인균인 Pseudomonas syringae pv. actinidiae는 유전적으로 구별되는 다섯 개의 biovar (1, 2, 3, 5, 6)로 나뉘어 진다. 그 중 biovar 3에 속하는 균주가 2008년 이래 전세계적으로 대유행을 일으키고 있다. 본 연구의 목표는 우리나라에서 분리된 biovar 3균주들의 집단 구조를 밝히고 그들의 기원을 추적하는데 있다. 13개의 우리나라 대표 균주와 2개의 중국 균주를 포함하는 15개 biovar 3 균주의 유전적 다양성을 RAPD-PCR로 평가하였다. RAPD 결과 연구에 사용된 균주들은 8개로 나누어져 이들을 subgroup I - VIII로 명명하였다. Subgroups II와 III은 중국 균주로 우리나라에서 발견되지 않았다. 따라서 우리나라 biovar 3 균주는 유전적으로 구별되는 6개의 subgroup (I, IV, V, VI, VII, VIII)으로 구성되어 있음을 알 수 있었다. New Zealand와 Europe균주에 특이적인 각각의 프라이머를 사용하여 PCR을 수행했을 때 subgroups V와 VI에 속하는 균주가 예상했던 DNA 절편을 증폭시켜 이들이 각각 두 지역에서 유입된 균주임을 시사하였다. 우리나라에서 처음 발견된 biovar 3 균주인 subgroup VIII에 특이적인 프라이머를 개발하여 조사한 결과 이 subgroup 균주는 처음 출현한 이후 더 이상 발견되지 않았다.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1280-1286
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• 2011

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at $3.6{\times}10^6$ CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above $10^5$/ml for approx. 10 days, after which cell numbers decreased to levels of $10^3$ CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. $1{\times}10^2$ CFU/ml was detected. The minimum detection level for quantitative PCR in this study was $1{\times}10^2$ to $1{\times}10^3$ CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

• 대한수의학회지
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• 제38권1호
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• pp.53-63
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• 1998

Total of 1085 swine sera (1996-1997) from nation-wide were tested for the presence of antibodies to influenza A virus. Fifty nine percent of the tested sera showed seropositive by HI test. Positive sera consisted of 24--- of H3, 15--- of H1, and 20--- of the sample had both antibodies, respectively. Sera collected from various region represented 7~27--- seropositivity to H1N1, 15~25--- to H3N2, respectively. Swine influenza field isolate from nasal swab was characterized antigenically and genetically to elucidate its relatedness with other known strains of influenza A virus. The study was focused on the HA gene which is related to pathogenecity and antigenic variability of the influenza virus. By RT-PCR using influenza A/H1N1 specific primers, influenza virus H1N1 specific DNA fragment was amplified from A/Swine/Iowa/15/30(H1N1), US field isolate but not in H3N2 strain. PCR products were sequenced by dideoxy chain termination method to determine nucleotide homology with other strains of influenza A virus. The US field isolate and A/Swine/Indiana/1726/88 strain had 97--- of nucleotide homology and 98--- of amino acid homology. Based on the results obtained from this experiment, the field isolate was genetically related to A/Swine/Indiana/1726/88 and had higher homology with A/Swine/Indiana/1726/88 than with classical swine influenza virus, A/Swine/Iowa/15/30. The field isolate had no amino acid changes at the antigenic site compare to that of the A/Swine/Indiana/1726/88. The proteolytic enzyme cleavage site between HA1 and HA2 had no alteration and the amino acid arginine was intact. There is no evidence has been found that the field isolate has genetic shift or genetic drift which might altered antigenic determinant.

• 식물조직배양학회지
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• 제27권1호
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• pp.7-12
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• 2000

저온관련 유전자인 BN115 gene과 표지유전자인 npt II gene을 함유하고 있는 Agrobacterium tumifacience GV 3101 균주를 이용하여 겨울상추품종인 청치마의 잎절편과 공동배양하는 방법으로 형질전환 시켰다. 상추의 잎절편을 Agrobacterium과 공동배양 후 MS 기본배지에 100 mg/L kanamycin, 500 mg/L carbenicillin, 0.1 mg/L NAA, 0.5 mg/L kinetin을 첨가한 선발배지에 치상하였는데, 치상 후 3-4주부터 절편체로부터 multiple shoot들이 생성되기 시작하였다. 선발배지에서 살아남은 선발체들은 1/2 MS배지에 100 mg/L kanamycin, 250 mg/L carbenicillin이 첨가된 발근배지로 옮겨졌다. 한편, 선발된 shoot들은 PCR반응을 이용하여 도입유전자의 삽입여부를 확인하였다. PCR 반응은 표지유전자인 nptII와 저온관련 유전자인 BN115 및 식물에 도입되지 않는 vir G 유전자를 각각 특이적으로 증폭하는 primer를 가지고 실시하였다. PCR 반응 결과 대조구로 쓰인 정상 상추식물체에서는 nptII와 BNl15유전자의 증폭을 볼 수 없는 반면에 형질전환체에서는 두 유전자 모두 PCR 증폭 산물을 확인할 수 있었다. 또한 확인된 식물체의 DNA에서는 vir G유전자가 발견되지 않아 이는 Agrobacterium의 혼입에 의한 결과가 아님을 다시 한번 증명하였다. 또한 선발된 형질전환체를 이용하여 Southern analysis와 RT-PCR을 실시한 결과 내한성 유전자가 상추 식물에 안정적으로 도입되어 발현됨을 확인하였다.

• International Journal of Industrial Entomology and Biomaterials
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• 제11권1호
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• pp.57-61
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• 2005

A strain of Bacillus thuringiensis that showed signifi­cantly high toxicity to Plutella xylostella was isolated from a dust sample collected from Chinese tobacco warehouse and characterized. The isolate named B. thuringiensis LY-99 was determined to belong to subsp. alesti (H3a3c) by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid and crystal protein patterns of the LY-99 were different from those of the reference strain, subsp. alesti. PCR analysis with specific primers revealed that this isolate contained abundant cry genes including crylAa, crylAc, crylB, crylD, crylE, crylF and cry2 genes, which was absolutely different from cry gene profile of the subsp. alesti. In addition, insecticidal activity of the LY-99 against P. xylostella larvae was about 44 times higher than that of the subsp. alesti.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.804-810
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• 1999

There is a possibility that carvone, a monoterpene from spearmint (Mentha spicata), could induce the bph degradative pathway and genes in Alcaligenes eutrophus H850, which is a known Gram-negative PCB degrader with a broad substrate specificity that was thoroughly investigated with Arthrobacter sp. BIB, a Gram-positive PCB degrader. The strains BIB and H850 were unable to utilize and grow on the plant terpene [(R)-(-)-carvone] (50ppm) to be recognized as a sole carbon source. Nevertheless, the carvone did induce 2,3-dihydroxybiphenyl 1,2-dioxygenase (encoded by bphC) in the strain B lB, as observed by a resting cell assay that monitors accumulation of a yellow meta ring fission product from 4,4'-dichlorobiphenyl (DCBp). The monoterpene, however, did not appear to induce the meta cleavage pathway in the strain H850. Instead, an assumption was made that the strain might be using an alternative pathway, probably the ortho-cleavage pathway. A reverse transcription (RT)-PCR system, utilizing primers designed from a conserved region of the bphC gene of Arthrobacter sp. M5, was employed to verify the occurrence of the alternative pathway. A successful amplification (182bp) of mRNA transcribed from the N-terminal region of the bphC gene was accomplished in H850 cells induced by carvone (50ppm) as well as in biphenyl-growth cells. It is, therefore, likely that H850 possesses a specific PCB degradation pathway and hence a different substrate specificity compared with B1B. This study will contribute to an elucidation of the dynamic aspects of PCB bioremediation in terms of roles played by PCB degraders and plant terpenes as natural inducer substrates that are ubiquitous and environmentally compatible.

• The Plant Pathology Journal
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• 제28권4호
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• pp.423-427
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• 2012

Pseudomonas syringae pv. actinidiae strains, the causal agents of bacterial canker on kiwifruit, were isolated from Korea and Italy in 2011. Among 87 isolates, a total of six representative strains, three from Korea and three from Italy, were identified on the basis of biochemical and physiological tests. Identities were confirmed by PCR using P. syringae pv. actinidiae-specific primers PsaF1/R2, which amplified a 280-bp DNA fragment. The strains isolated from Korea in this study displayed BOX-PCR patterns similar to those isolated from Italy but different from those isolated previously in Korea or the pathotype P. syringae pv. actinidiae strain. The effector hopA1 and hopH1 genes, which are known to be present in strains isolated recently from France and Italy, were also present in P. syringae pv. actinidiae strains, SYS1, SYS2 and SYS4, isolated from Korea in this work. However, no amplicons of the expected size were obtained from strains previously isolated from Korea and Japan. In addition, the Korean strains isolated in this work belonged to haplotype I for the cts gene identical to those strains isolated from recent outbreaks in Italy. These results suggest that P. syringae pv. actinidiae strains isolated from Korea and examined in this work are a new type of strain similar to those found from recent outbreaks in Italy. This is the first report on the occurrence of cts haplotype I strains of P. syringae pv. actinidiae affecting kiwifruit plants in Korea.