• Journal of Pharmaceutical Investigation
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• 제31권3호
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• pp.185-190
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• 2001

Human papillomavirus (HPV) infection is known to cause cervical cancers. Human papillomavirus-like particles (VLP) have been studied as preventive vaccines of cervical cancers. To develop VLP as a therapeutic gene carrier, we studied the method to encapsulate cytokine genes in virus-like particles. HPV type 16 capsid L1 genes were amplified by polymerase chain reaction and cloned into T vector. L1 gene was then inserted into baculovirus transfer vector. The clone of baculovirus encoding L1 gene was isolated and used to express L1 protein in Sf 21 insect cells. VLP were purified by CsCl density gradient and ultracentrifugation. VLP were disassembled to capsomer units by treatment of a reducing agent. Given that interleukin-2 (IL-2) genes have been used in anticancer gene therapy and as a molecular adjuvant, IL-2 cytokine plasmids were chosen as a model gene. IL-2 plasmids were incubated with the disassembled capsomer suspension. To reassemble the particles, the mixture of capsomers and cytokine plasmids was dialyzed. The disassembly and reassembly of VLP were confirmed by transmission electron microscopy. The entrapment of cytokine plasmids in reassembled VLP was tested by the stability of plasmids against DNase I. After treatment of reassembled virus-like particles with DNase I, discrete IL-2 DNA band was observed. Our results indicate that IL-2 cytokine plasmid (3.5 kb size) can be encapsulated in the virus-like particles, suggesting the potential of VLP as a gene delivery system. Moreover, VLP containing the adjuvant cytokine plasmids might function as more effective subunit vaccines.

• 한국식품영양과학회지
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• 제39권4호
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• pp.595-601
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• 2010

본 연구는 국내 주요 식중독 원인균인 Staphylococcus aureus, Salmonella enterica subsp., Vibrio parahaemolyticus를 동시에 검출 및 동정할 수 있는 simultaneous multiplex PCR방법을 개발하고자 하였다. S. aureus의 23s rRNA 유전자(482 bp), V. Parahaemolyticus의 toxR 유전자(368 bp), S. enterica subsp.의 invA 유전자(284 bp)를 특이적으로 검출 및 동정할 수 있는 3개 primer set 즉, STA-5F/STA-5R, ToxR-F/ToxR-R, 139/141을 구축하였으며, 그 결과 정제되어진 각 식중독 원인균의 genomic DNA를 template로 하여 세 균주 모두 10 pg까지 다중동시검출이 가능하였다. 생균수(CFU)와 상응되는 검출한계 결과로써 $10^1\sim10^2$ CFU/reaction의 검출한계를 보였으며 이는 즉, S. aureus $6.0\times10^4$ CFU/mL, S. enterica subsp. $9.5\times10^4$ CFU/mL, V. parahaemolyticus $6.1\times10^5$ CFU/mL의 검출한계를 나타내었다. 균체회수부터 agarose gel 상에서 검출 및 동정까지 3~4 hr의 시간 소요로 single tube 반응으로 세 식중독 원인균의 다중동시검출이 가능하였다. 또한 추가적인 연구를 통하여 세 식중독 원인균주의 검출을 위한 향상된 민감도를 가지는 multiplex PCR법 및 real time PCR을 이용한 다중동시검출법 개발을 위한 기초자료로서 활용 가능할 것이라 사료된다.

• Journal of Animal Science and Technology
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• 제47권3호
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• pp.317-324
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• 2005

A method for sex determination of pigs was examined using polymerase chain reaction(PCR). Sex determining region Y(SRY) gene encoded on Y chromosome plays a key role for primary male development. Zinc finger X-Y(ZFX-ZFY) gene, one of the X-V homology gene group was found on the X and Y chromosomes, respectively, We tested for molecular sexing by amplification patterns of SRY and ZF genes. Genomic DNAs from various resources including porcine hairs and semen collected from domestic pig breeds and native pigs was used for PCR assay of each gene. The amplified products for porcine SRY gene were yielded only in males but not in females. On the other hand, two differential patterns were observed in amplification of ZF gene reflecting the chromosomal dimorphism by a length polymorphism between X and Y chromosomes. Of both, a common band was detected in all individuals tested so that this band might be amplified from ZFX gene as a PCR template, but another is specific for males indicated that from ZFY. The result of PCR assay provides identical information to that from investigation of phenotypic genders of the pigs tested. We suggest that this PCR strategy to determine porcine sexes using comparison of the amplification patterns of the SRY gene specific for Y chromosome and the dimorphic ZF gene between X and Y chromosomes may be a rapid and precise method for discrimination of two sexes and applied to DNA analysis of small samples such as embryonic blastomere, semen, and hairs.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.91-101
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• 2018

The aim of this study was to transfer the 18.5 kb gene clusters coding for 17 genes from Lactobacillus rhamnosus to Lactococcus lactis subsp. cremoris MG1363 in order to determine the effect of host on exopolysaccharide (EPS) production and to provide a model for studying the phosphorylation of proteins which are proposed to be involved in EPS polymerization. Lactobacillus rhamnosus RW-9595M and ATCC 9595 have 99% identical operons coding for EPS biosynthesis, produced different amounts of EPS (543 vs 108 mg/l). L. lactis subsp. cremoris MG1363 transformed with the operons from RW-9595M and ATCC 9595 respectively, produced 326 and 302 mg/l EPS in M17 containing 0.5% glucose. The tyrosine protein kinase transmembrane modulator (Wzd) was proposed to participate in regulating chain elongation of EPS polymers by interacting with the tyrosine protein kinase Wze. While Wzd was found in phosphorylated form in the presence of the phosphorylated kinase (Wze), no phosphorylated proteins were detected when all nine tyrosines of Wzd were mutated to phenylalanine. Lactococcus lactis subsp. cremoris could produce higher amounts of EPS than other EPS-producing lactococci when expressing genes from L. rhamnosus. Phosphorylated Wzd was essential for the phosphorylation of Wze when expressed in vivo.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.735-738
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• 2001

Metabolically engineered Escherichia coli strains harboring a plasmid containing a novel artificial polyhydroxyalkanoate (PHA) operon consisting of the Aeromonas PHA biosynthesis related genes and Ralstonia eutropha reductase gene were developed for the production of poly(3-hydroxybutyrate-co-hydroxyhexanoate) [P(3HB-co-3HHx)] from dodecanoic acid. By applying stepwise reduction of dissolved oxygen concentration (DOC) during the fermentation, the final dry cell weight, PHA concentration, and PHA content of 79 g/L, 21.5 g/L, and 27.2 wt%, respectively, were obtained in 40.8 h, which resulted in the PHA productivity of 0.53 g/L/h. The 3HHx fraction slowly increased during the fed-batch culture to reach a final value of 10.8 mol%. The 3HHx fraction in the copolymer could be increased by three fold when the Aeromonas hydrophila orfl gene was co-expressed with the PHA biosynthesis genes.

• IMMUNE NETWORK
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• 제3권3호
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• pp.227-234
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• 2003

Background: Immunoglobulin (Ig) light chain repertoire has been implicated as a critical determinant in regulation of autoreactive B cells and production of pathogenic anti-DNA antibodies in systemic lupus erythematosus (SLE). We analyzed the impact of Ig ${\lambda}$ chain repertoire on development of autoimmunity in patients with SLE. Methods: We obtained genomic DNA from individual peripheral CD19+ B cells of 3 untreated active SLE patients, and amplified $V{\lambda}$ rearrangements from each single cell by polymerase chain reaction. Results: A total number of 208 $V{\lambda}J{\lambda}$ rearrangements were analyzed. Analyzed sequences included 158 productive rearrangements and 50 nonproductive rearrangements. The differences in $V{\lambda}$ gene usage in the productive and nonproductive repertoire of SLE patients were found compared to the non-autoimmune individuals. $V{\lambda}$ gene, 9A was significantly overrepresented in nonproducative repertoire of SLE patients (P=0.016). In the productive repertoire, $V{\lambda}$ genes, 3L and 1E were found more often in the SLE patients (P=0.001, P=0.043). When the productive and the nonproductive repertoires were compared, 9A was found significantly less in the productive repertoire in the SLE patients (P=0.000). There were no significant differences in the $J{\lambda}$ gene usage between SLE patients and non-autoimmune individuals, but $J{\lambda}2/3$ gene was the most frequently used in SLE, whereas $J{\lambda}7$ gene was the most frequently used in the normal subjects. In the productive SLE $V{\lambda}$ repertoire, 9.4% of the total sequences employed identical CDR3. It was particularly striking to find 7 identical versions of the 1G-$J{\lambda}2/3$ $V{\lambda}J{\lambda}$ rearrangements from one patient and 3 of the same sequence from another patient. Notably, identical $V{\lambda}$ junctions in the SLE patients utilized significantly more homologous joining compared to $V{\lambda}$ junctions of the normal adults (P=0.044). Conclusion: These data demonstrate regulation of ${\lambda}$ light chain expression in the SLE patients by selection of unique $V{\lambda}$ genes. Also, biased selection and clonal expansion of particular $V{\lambda}$ rearrangements are apparent in the SLE ${\lambda}$ repertoire.

• Journal of Plant Biotechnology
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• 제1권2호
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• pp.85-90
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• 1999

In order to protect fungal diseases, leaf disc explants of Solanum tuberosum cultivar, Belchip, was infected with an Agrobacterium MP90 strain containing chimeric gene construct, consisting of antibiotic resistance and chitinase gene driven by the CaMV 35S promoter, for transformation. Regenerated multiple shoots were selected on a medium containing kanamycin and carbenicillin after exposure to Agrobacterium. The presence and integration of the npt II and chitinase gene were confirmed by polymerase chain reaction(PCR). Northern blot analysis indicated that the genes coding for the enzyme could be expressed in potato plants. The chitinase activity of transgenic potato plants was higher than the control potato.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.845-855
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• 2015

The present study describes the gene cloning and high-level expression of an alkaline and thermostable lipase gene from Trichosporon coremiiforme V3. Nucleotide analysis revealed that this lipase gene has an open reading frame of 1,692 bp without any introns, encoding a protein of 563 amino acid residues. The lipase gene without its signal sequence was cloned into plasmid pPICZαA and overexpressed in Pichia pastoris X33. The maximum lipase activity of recombinant lipase was 5,000 U/ml, which was obtained in fed-batch cultivation after 168 h induction with methanol in a 50 L bioreactor. The purified lipase showed high temperature tolerance, and being stable at 60℃ and kept 45% enzyme activity after 1 h incubation at 70℃. The stability, effects of metal ions and other reagents were also determined. The chain length specificity of the recombinant lipase showed high activity toward triolein (C18:1) and tripalmitin (C16:0).

• 식물조직배양학회지
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• 제28권2호
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• pp.87-90
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• 2001

배추 유래의 cytosolic glutathione reductase 유전자 (BcGR1)의 지속적 발현과 형질전환 식물체의 oxidative stress에 대한 내성과의 관계를 분석하기 위하여, BcGR1 유전자를 CaMV 35S promoter의 하류에 연결한 다음, 담배에 형질전환하였다. PCR 및 Southern blot 분석을 통하여 BcGR1 유전자가 정상적으로 삽입된 32 계통의 T$_{0}$ 식물체를 선발하였다. Northern blot 분석 결과, 도입된 유전자가 형질 전환 식물체 내에서 항상적으로 발현된다는 것을 확인하였으며, 도입 유전자의 copy number와 발현량 사이에는 정의 상관관계를 보이지 않았다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권2호
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• pp.117-127
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• 2001

The high-level expression of a human single chain urokinase-type plasminogen activator (scu-PA) was achieved by employing a methotrexate (MTX)-dependent gene amplification system in Chinese hamster ovary (CHO) cells. By cotransfecting and coamplifying a scu-PA expression plasmid and dihydrofolate reductase (DHFR) minigene, several scu-PA expressing CHO cell lines were selected and gene-amplified. These recombinant cell lines, NGpUKs, secreted a completely processed scu-PA of 54 kD and up to 60mg/L was accumulated in the culture medium when they were adapted to an optimal MTX concentration. Over 95% of the scu-PA expressed was secreted in the culture medium and identified having the proper function of a plasminogen activator when activated by plasmin. Based on a genomic Southern analysis, a representative subclone, MGpUK-5, exhibited MTX-dependent scu-PA gene amplification, plus the initial single-copy gene of scu-PA eventually turned into about 150 copies of the amplified gene of scu-PA after gradual adaptation to 2.0$\mu$M of MTX. Meanwhile, the transcripts kof the scu-PA gene increased, although -early saturation of transcription was identified at 0.1$\mu$M of MTX. The scu-PA production by the MGpUK-5 subclone also increased relative to the gene amplification and increased transcripts, however, the relationship was not linearly proportional. Accordingly, since the MGpUK cell lines expressed elevated levels of enzymatically active scu-PA, these cell lines could be applied to the largescale production of scu-PA.