• International Journal of Industrial Entomology and Biomaterials
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• 제7권2호
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• pp.197-201
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• 2003

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni Tn 5B1-4 cells and Bombyx mori BmN cells, respectively. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 and BmN cells was secreted into the medium. BmN cells are relatively lower in maximum cell growth and recombinant endostatin production than Tn 5B 1-4 cells. Recombinant endostatin was also purified to homogeneity using a simple one-step ${Ni^2+}$ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition $({ED_50})$ for recombinant endostatin was approximately 0.35 ${\mu}g$/ml.

• Applied Biological Chemistry
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• 제44권1호
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• pp.1-6
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• 2001

본 연구에서는 Ttichoplusia ni 세포의 apoptosis 유도 및 억제 현상의 기초연구를 수행하였다. Apoptosis 유도제로 알려진 hygromycin B에 의한 세포 성장 저해는 $200\;{\mu}/ml$의 수준에서부터 나타났고, $400\;{\mu}/ml$ hygromycin B를 처리한 세포에서는 배양 후 2일부터 DNA가 분절되어지는 것을 확인할 수 있었다. 그러나 dexamethasone과 sodium butyrate를 첨가시 세포성장은 저해되었지만 DNA 분절현상이 보이지 않아 apoptosi의 유발여부를 확인할 수 없었다. 그리고 caspase 기능억제제의 apoptosis 지연효과를 보기 위해 $200\;{\mu}/ml$ hygromycin B로 apoptosis를 유발한 상태에서 Ac-DEVD-CHO를 첨가하여 세포성장을 비교해 본 결과 이 저해제에 의해 약 36%정도 apoptosis가 억제되었음을 확인하였다. N-acetylcysteine의 경우도 apoptosis지연 효과가 있었다. Bcl_계에 속하는 anti-apoptotic 유전자의 발현연구로서 apoptosis 저해 단백질인 bcl-2 유전자를 곤충세포에 형질전환시킨 후 이 단백질이 한시적으로 발현되는 것을 western blot분석법으로 확인하였으며 apoptosis가 지연된 곤충세포주의 개발이 가능하다는 결론을 보였다.

• 생명과학회지
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• 제24권1호
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• pp.86-91
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• 2014

Trichoplusia ni (T. ni) 세포는 사람 당 수송체를 이성질적으로 많은 양 생산하려 할 때 유용하게 사용되는, baculovirus 발현 시스템의 숙주세포로서 이용된다. 그러나 T. ni 세포에 존재하는 내재된 당 수송체의 높은 활동은, 발현된 외재적 당 수송체의 수송활성과 같은 직접적 증거 제시에 장애가 된다. 뿐만 아니라 곤충세포에 내재하는 당 수송체계의 특성에 대해서는 밝혀진 바가 거의 없다. 그래서 본 연구에서는 baculovirus 발현 시스템을 보다 잘 활용하기 위해 T. ni 세포의 2dGlc기질 수송에 D-fructose가 미치는 영향을 살펴 보았으며, T. ni 세포에 발현된 사람 당 수송체의 생물학적 활성을 보다 용이하게 검증하기 위해 발현된 수송체를 [$^3H$] cytochalasin B를 이용하여 photolabelling 하였다. 우선 감염되지 않은 세포와 recombinant AcMPV-GTL 감염시킨 T. ni 세포의 2dGlc uptake를 300 mM D-fructose가 있을 때와 없을 때, 그리고 $20{\mu}M$ cytochalasin B가 있을 때와 없을 때의 상황에서 살펴보았다. 감염되지 않은 세포에서의 육탄당 uptake는 D-fructose에 의해 강력하게 억제 되었으나 cytochalasin B에 의해서는 단지 미미한 억제 효과만을 보여주었다. 흥미롭게도 AcMPV-GTL 바이러스 감염된 T. ni 세포에서는 비록 2dGlc uptake율은 감염되지 않은 세포와 비교해 다소 낮았지만 육탄당 수송 억제 반응은 근본적으로 동일함을 보여 주었다. 또한 [$^3H$] cytochalasin B를 이용한 발현단백질 photolabelling에서는, L-glucose가 존재하는 상황 하에만 하나의 날카롭게 표지된 peak가, 바이러스 감염된 세포에서 관찰되었다. 감염되지 않은 세포에서는 이러한 peak는 관찰되지 않았다. 게다가 D-glucose 존재 하에서는 발현된 단백질의 photolabelling이 완전히 억제되어짐을 보여주어, labelling의 입체선택성(stereoselectivity)을 입증하였다.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2002년도 Join Meetings of Korean Society of Sericultural Science and Japanse Society of Sericultural Science
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• pp.65-65
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• 2002

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.739-744
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• 2007

Insect cell lines and the control of infection for obtaining the maximum amount of polyhedrin-Cry1Ac-polyhedrin fusion protein from Bactrus in monolayer and suspension culture systems were tested. Growth rates of the Trichoplusia ni(High-Five) cell line in both culture systems were better than the other insect cell lines, Spodoptera frugiferda(Sf-9, Sf-21), Trichoplusia ni(Tn5), and Spodoptera exigua(Se301). The expression of the fusion protein in a monolayer culture showed that Se301 cells were 2.3-4.8 times more productive on a per cell basis than the other cell lines. However, in suspension culture, only High-Five cells were productive. High-Five cells infected with Bactrus at a multiplicity of infection(MOI) of 5 and a cell density of $3.0{\times}10^5$ cells per ml were more productive than the other infection condition in a suspension culture suitable for a large-scale production of baculovirus. In conclusion, for the large-scale production of Bactrus in vitro, High-Five cells showing good growth and high productivity are suitable.

• 한국양봉학회지
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• 제34권2호
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• pp.137-140
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• 2019

Although honeybees (Apis mellifera) are crucial for maintenance of the ecosystem, population of honeybee has been steadily decreasing due to diseases including nosemosis. Nosemosis is a disease caused by Nosema ceranae and is now considered as a major threat to honeybees. N. ceranae is a microsporidian that stays in form of spore even before the infection, which makes it harder to control than other pathogens. People are now aware of this parasite, however, cure and preventive candidates for nosemosis are hardly found until today. In this study, in vitro experiment of Lespedeza cuneata treatment to prevent nosemosis were done using Trichoplusia ni cell line, BTI-TN5B1-4. Normal T. ni cells exhibited round shape without abnormal size. On the other hand, when N. ceranae were treated, cells deteriorated and some cells abnormally enlarged due to N. ceranae infection. Interestingly, treatment of T. ni cells with L. cuneate extract protected abnormal cell shape induced by N. ceranae infection to normal shape. Some N. ceranae spores were observed outside of the cells. Effective concentration range for N. ceranae control were experimented. Lowest concentration which can control nosemosis were 50 ㎍/mL. When the concentration of L. cuneata extract was exceeded 200 ㎍/mL, cytotoxicity started to show up.

• BMB Reports
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• 제31권5호
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• pp.453-458
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• 1998

Human thrombopoietin (hTPO) was expressed to high levels in insect cells using the baculovirus expression system. Full-length hTPO cDNA containing a native signal peptide sequence was amplified by PCR from a human fetal liver cDNA library and cloned into the Autographa californica nuclear polyhedrosis virus (AcNPV) expression vector. Immunoblot analysis with antiserum against hTPO indicated that an approximately 55 kDa protein was produced in recombinant AcNPV infected insect cells. Recombinant hTPO was produced 4-fold higher in Trichoplusia ni (Tn5) cells than in Spodoptera frugiperda (Sf9) cells. with most of the hTPO produced in Tn5 cells secreted into the culture medium. Addition of tunicamycin in the culture medium resulted in the reduction of the size of hTPO to 35-38 kDa, and most of the protein remained within the cell. These results suggest that N-glycosylation of hTPO is required for the secretion of the protein into the culture medium in insect cells. hTPO produced in insect cells induced proliferation and maturation of megakaryocyte progenitors, indicating that it is in a biologically active form.

• Journal of Microbiology
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• 제45권2호
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• pp.133-138
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• 2007

This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via ${\beta}-galactosidase$ expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.383-391
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• 2008

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed in mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennarytype complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using a pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of Nacetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using $UDP-^{14}C-Gal\;and\;CMP-^{14}C-Sia$ as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the ed blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.