• Journal of Microbiology and Biotechnology
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• 제29권10호
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• pp.1644-1655
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• 2019

Saccharomyces cerevisiae (S. cerevisiae) and glucagon-like peptide-2 (GLP-2) have been employed to improve the intestinal development of weaned animals. The goal of this study was to determine whether either exogenous S. cerevisiae or GLP-2 elicits major effects on fecal microbiotas and cytokine responses in weaned piglets. Ninety-six piglets weaned at 26 days were assigned to one of four groups: 1) Basal diet (Control), 2) empty vector-harboring S. cerevisiae (EV-SC), 3) GLP-2-expressing S. cerevisiae (GLP2-SC), and 4) recombinant human GLP-2 (rh-GLP2). At the start of the post-weaning period (day 0), and at day 28, fecal samples were collected to assess the bacterial communities via sequencing the V1-V2 region of the 16S-rRNA gene, and piglets' blood was also sampled to measure cytokine responses (i.e., IL-$1{\beta}$, TNF-${\alpha}$, and IFN-${\gamma}$). This study revealed that, on the one hand, although S. cerevisiae supplementation did not significantly alter the growth of weaned piglets, it induced increases in the relative abundances of two core genera (Ruminococcaceae_norank and Erysipelotrichaceae_norank) and decreases in the relative abundances of two other core genera (Lachnospiraceae_norank and Clostridiale_norank) and cytokine levels (IL-$1{\beta}$ and TNF-${\alpha}$) (p < 0.05, Control vs EV-SC; p < 0.05, rh-GLP2 vs GLP2-SC). On the other hand, GLP-2 supplementation had no significant influence on fecal bacterial communities and cytokine levels, but it produced better body weight and average daily gain (p < 0.05, Control vs EV-SC; p < 0.05, rh-GLP2 vs GLP2-SC). Therefore, altered fecal microbiotas and cytokine response effects in weaned piglets were due to S. cerevisiae rather than GLP-2.

• Journal of Korean Neurosurgical Society
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• 제65권6호
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• pp.825-833
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• 2022

Objective : ABM/P-15 (anorganic bone matrix/15-amino acid peptide fragment) is a commercially available synthetically manufactured P-15 collagen peptide fragment, that is adsorbed on ABM. This study was done to investigate the efficacy of ABM/P-15 in achieving fusion in the lumbar spine and comparing it with that of recombinant bone morphogenic protein-2 (rhBMP-2) and demineralized bone matrix (DBM). Methods : A retrospective observational study of prospectively collected data of 140 patients who underwent lumbar spinal fusion surgeries in a single specialty spine hospital between 2016 and 2020, with a minimum 6-month follow-up was conducted. Based on the material used for the augmentation of the bone graft at the fusion site, the patients were divided into three categories namely ABM/P-15, rhBMP-2, and DBM group. Results : ABM/P-15, rhBMP-2, and DBM were used in 46, 44, and 50 patients, respectively. Patient characteristics like age, gender, bone mineral density, smoking history, and presence of diabetes mellitus were comparable amongst the three groups. Average follow-up was 16.0±5.2, 17.9±9.8, and 26.2±14.9 months, respectively in ABM/P-15, rhBMP-2, and DBM groups. The fusion was achieved in 97.9%, 93.2%, and 98% patients while the average time-to-union was 4.05±2.01, 10±4.28, and 9.44±3.49 months (p<0.001), respectively for ABM/P-15, rhBMP-2, and DBM groups. The average pre-operative Visual analogue scale score was 6.93±2.42, 7.14±1.97, 7.01±2.14 (p=0.900) for ABM/P-15, rhBMP-2 and DBM groups, respectively, which reduced to 1.02±0.80, 1.21±0.96, and 0.54±0.70 (p=0.112), respectively at the last follow up. Pre-operative Oswestry disability index scores were 52.7±18.02, 55.4±16.8, and 53.56±19.6 (p=0.751) in ABM/P-15, rhBMP-2, and DBM groups, which post-operatively reduced to 33.77±15.52, 39.42±16.47, and 38.3±15.89 (p=0.412) and further to 15.74±8.3, 17.41±10.45, and 16.76±9.81 (p=0.603), respectively at the last follow-up. Conclusion : ABM/P-15 appears to achieve union significantly earlier than rhBMP-2 and DBM in lumbar spinal fusion cases while maintaining a comparable clinical and complication profile.

• 한국가금학회지
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• 제38권4호
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• pp.323-330
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• 2011

닭의 단일 클론 항체인 13C8 항체는 조류의 콕시듐증을 유발하는 것으로 알려진 Eimeria acervulina의 포자충(sporozoites) 항원에 결합하는 닭 항체이다. 그러나 닭 하이브리도마 유전자의 불안정성 때문에 분비되는 항체의 생산량이 낮은 단점이 있다. 이러한 단점을 보완하기 위해 hybridoma로 부터 항체의 중사슬 가변 부위(VH)유전자와 경사슬 가변 부위(VL) 유전자를 증폭하여 linker peptide로 연결해준 재조합 ScFv 항체 유전자를 구축하고, E. coli를 형질 전환시켜 재조합 단백질로 발현 정제하였다. ELISA 분석 결과 재조합 13C8 ScFv 항체는 세 종류의 Eimeria spp.에 대해 모두 항원 결합력을 나타내었으며, 염기서열 분석을 수행하여 germline sequence와 비교한 결과 닭 항체유전자의 다양성(diversity)은 pseudogene들의 gene conversion 기작에 의해 이루어짐을 제시해 주었다.

• Journal of Microbiology and Biotechnology
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• 제19권5호
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• pp.502-510
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• 2009

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmci reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked inmmunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active aligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

• 미생물학회지
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• 제42권2호
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• pp.142-148
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• 2006

본 연구는 다양한 생리활성을 가지고 있는 chitosan oligosaccharides를 효소적 방법으로 생산하기 위한 기초 연구로서, 전통발효식품인 메주에서 chitosan 분해능이 우수한 균주를 분리하였다. 분리한 균주를 형태학적, 생화학적 및 분자생물학적 방법을 이용하여 동정한 결과, Bacillus amyloliquefaciene MJ-1으로 명명하였다. B. amyloliquefaciene MJ-1으로 부터 chitosanase 유전자를 포함하는 1,049 bp DNA 단편을 클로닝하였으며, chitosanase 유전자는 825 염기로서 274 개의 아미노산으로 구성되어 있었고, 예상 분자랸은 30.9 kDa이었다. 클로링한 chitosanase의 homology search 결과, glucoside hydrolase family 46에 속하는 chitosanase로 추정되었다. B. amyloliquefaciene MJ-1 chitosanase 유전자를 E. coli BLR (DE3)에 도입하였으며, 1 mM의 IPTG로 chitosanase 과잉 발현을 유도하고 정제한 후, pH및 온도에 대한 특성을 조사하였다. 효소 활성의 최적 온도는 $60^{\circ}C$이었으며, $80^{\circ}C$에서도 75%의 활성을 나타내었으므로 내열성을 가진 효소로 추정되었다. 한편, 최적 pH는 5.0 이었으며, pH $5{\sim}7$사이에서 80% 이상의 높은 활성을 유지하였다.

• IMMUNE NETWORK
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• 제5권2호
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• pp.55-67
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• 2005

Cancer vaccine is an active immunotherapy to stimulate the immune system to mount a response against the tumor specific antigen. Working as a stimulant to the body's own immune system, cancer vaccines help the body recognize and destroy targeted cancers and may help to shrink advanced tumors. Research is currently underway to develop therapeutic cancer vaccines. It is also possible to develop prophylactic vaccines in the future. The whole cell approach to eradicate cancer has used whole cancer cells to make vaccine. In an early stage of this approach, whole cell lysate or a mixture of immunoadjuvant and inactivated cancer cells has been used. Improved vaccines are being developed that utilize cytokines or costimulatory molecules to mount an attack against cancer cells. In case of melanoma, these vaccines are expected to have a therapeutic effect of vaccine. Furthermore, it is attempting to treat stomach cancer, colorectal cancer, pancreatic cancer, and prostate cancer. Other vaccines are being developing that are peptide vaccine, recombinant vaccine and dendritic cell vaccine. Out of them, reintroduction of antigen-specific dendritic cells into patient and DNA vaccine are mostly being conducted. Currently, research and development efforts are underway to develop therapeutic cancer vaccine such as DNA vaccine for the treatment of multiple forms of cancers.

• Parasites, Hosts and Diseases
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• 제40권3호
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• pp.131-138
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• 2002

The cyclophilins (Cyps) are family members of proteins that exhibit peptidylprolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosprin A (CsA) in varying degrees. During the process of random sequencing of a cDNA library made from Giardia intestinalis WB strain, the cyclophilin gene (gicypl) was isolated. An open reading frame of gicyp1 gene was 576 nucleotides, which corresponded to a translation product of 176 amino acids (Gicypl). The identity with other Cyps was about 58-71%. The 13 residues that constituted the CsA binding site of human cyclophilin were also detected in the amino acid sequence of Gicypl, including tryptophan residue essential for the drug binding. The single copy of the gicypl gene was detected in the G. intestinalis chromosome by southern hybridization analysis. Recombinant Gicyp 1 protein clearly accelerated the rate of cis ${\rightarrow}$ trans isomerization of the peptide substrate and the catalysis was completely inhibited by the addition of $0.5{\;}{\mu}M$ CsA.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.29-36
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• 2007

The xynA gene encoding the xylanase A of Paenibacillus sp. DG-22 was isolated with a DNA probe obtained by PCR amplification, using degenerated primers deduced from the amino acid residues of the known N-terminal region of the purified enzyme and the conserved region in the family 11 xylanases. The positive clones were screened on the LB agar plates supplemented with xylan, by the Congo-red staining method. The xynA gene consists of a 630-bp open reading frame encoding a protein of 210 amino acids, and the XynA preprotein contains a 28-residues signal peptide whose cleavage yields a l82-residues mature protein of a calculated molecular weight of 20,000Da and pI value of 8.77. The cloned DNA fragment also has another ORF of 873 nucleotides that showed 76% identity to the putative transcriptional activator of Bacillus halodurans C-125. Most of the xylanase activity was found in the periplasmic space of E. coli. The xynA gene was subcloned into pQE60 expression vector to fuse with six histidine-tag. The recombinant xylanase A was purified by heating and immobilized metal affinity chromatography. The optimum pH and temperature of the purified enzyme were 6.0 and $60^{\circ}C$, respectively. This histidine-tagged xylanase A was less thermostable than the native enzyme.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.591-597
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• 2003

Human urokinase kringle domain, sharing homology with angiostatin kringles, has been shown to be an inhibitor of angiogenesis, which can be used for the treatment of cancer, rheumatoid arthritis, psoriasis, and retinopathy. Here, the expression of the kringle domain of urokinase (UK1) as a secreted protein in high levels is reported. UK1 was expressed in the methylotrophic yeast Pichia pastoris GS115 by fusion of the cDNA spanning from Ser47 to Lys135 to the secretion signal sequence of ${\alpha}-factor$ prepro-peptide. In a flask culture, the secreted UK1 reached about 1 g/l level after 120h of methanol induction and was purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the purified UK1 revealed that it was cleaved at the Ste13 signal cleavage site. The molecular mass of UK1 was determined to be 10,297.01 Da. It was also confirmed that the purified UK1 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, or epidermal growth factor, in a dose-dependent manner. These results suggest that a P. pastoris sytem can be employed to obtain large amounts of soluble and active UK1.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.191-193
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• 2000

B. macerans 유래의 CGTase를 yeast surface display기술을 이용하여 S. cerevisiae의 표면에 발현된 것을 halo-test와 immunofluorescence microscopy와 flow cytometry를 통하여 확인하였다. 재조합 효모는 효소의 cyclization작용을 저해하고 CD의 분해작용을 촉진하는 glucose와 maltose를 제거하는 발효공정과 표면 발현된 CGTase의 cyclization 공정을 동시에 수행할 수 있어 CD의 생산, 분리공정을 효율적으로 개선하였다.