• International Journal of Industrial Entomology and Biomaterials
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• 제3권1호
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• pp.13-18
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• 2001

The role of polyhedrin in the polyhedra production in baculovirus Autograha californica Nucelopolyhedro-sisvirus (AcNPV) was studied by over-expression of AcNPV polyhedrin or heterologous polyhedrin from Bombyx mori (Bm) NPV or Spodoptera exigua (Se) NPV. The transfer vectors containing additional polyhedrin from AcNPV, BmNPV, or SeNPV were constructed and cotransfected with bacmid bApGOZA into Sf9 cells. The resulting recombinants, designated as vApAcPol, vApBmPol, and vApSePol were tonstructed, and the polyhedra production of the recombinant was characterized. All of the recombinants produced polyhedra in the nucleus, and the polyhedrin was over-expressed. Among three recombinants, vApAcPol and vApBmPol were discriminated by their larger polyhedra size than that of wild type AcNPV, and vApSePol also produced larger polyhedra than wild type SeNPV polyhedra.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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• pp.142-146
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• 2003

Three insect cell lines, Sf9, Sf21 and Tn5Bl-4, and four different kinds of serum free media (SFM), Sf 900 II, EX-CELL 420, EX-CELL 405 and Express Five, were used to compare the nutrient consumption, byproduct formation, production of recombinant protein and protease activity in suspension cultures. The Sf 900 II SFM was a ppropriate for the cell growth and protein production of the Sf9 and Sf21 cell lines. When the Tn5Bl-4 cell line was grown in the Express Five SFM, the specific growth rate was 1.6 fold higher than those of either the Sf9 or Sf21 cell lines. The glucose and glutamine consumption rates per cells, were 4 and 2.3 times higher than those of the Sf9 cell line, respectively. The overall yield coefficients of the lactate and ammoniumion were 2.8 and 1.5 times higher compared to those of the Sf9 cell line. respectively. The maximum specific ${\beta}$-galactosidase production rate was 4.5 fold that of the Sf9 cell line, a 3 times higher protease activity per cell.

• Archives of Pharmacal Research
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• 제20권5호
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• pp.396-403
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• 1997

In order to direct the form of the immune response in an antigen-specific manner, we constructed a fusion protein (OVA/IL12) that contained the T cell-dependent antigen, ovalbumin (OVA), covalently linked to murine interleukin-12 (IL-12). The OVA/IL12 protein was produced in a baculovirus expression system and was purified by anti-OVA immunoaffinity chromatography. The purified OVA/ILI2 protein displayed potent IL-12 bioactivity in an IL-12 proliferation assay. BALB/c mice immunized with the OVA/IL12 protein produced increased quantities of anti-OVA IgG2a antibody compared with mice immunized with recombinant OVA alone. Lymph node cells from the immunized mice with the OVA/IL12 protein produced large amounts of IFN-,Y when restimulated in vitro with OVA, while those from mice immunized with the OVA protein produced little or no IFN-.gamma.. In contrast, immunization with a mixture of OVA and free recombinant IL-12 also induced IFN-.gamma. production, which was not OVA-specific. These studies indicate that the OVA/IL12 fusion protein can induce OVA-specific, Th1-dominated immune responses, and that the covalent linkage of OVA and IL-12 confines the effect of IL-12 to OVA-specific cells.

• 대한의생명과학회지
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• 제11권1호
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• pp.57-61
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• 2005

The number of sites at which a protein can be readily cleaved by a proteolytic enzyme is greatly influenced by its three-dimensional structure. For native, properly-folded proteins both the rate of cleavage and number of sites at which cleavage takes place are usually much less than for the denatured protein. In order to compare the tertiary structure of recombinant HepG2 type glucose transporter with that of its native counterpart in the erythrocyte, the pattern of tryptic cleavage of the protein expressed in insect cell membranes was therefore examined. After 30 minutes digestion, a fragment of approximate Mr 19,000-21,000 was generated. In addition to this, there were two less intensely stained fragments of apparent Mr 28,000 and 17,000. The pattern of labelling was similar up to 2 hours of digestion. However, the fragments of Mr 19,000-21,000 and Mr 17,000 were no longer detectable after 4 hours digestion. The observation of a very similar pattern of fragments yielded by tryptic digestion of the HepG2 type transporter expressed in insect cells suggests that the recombinant protein exhibits a tertiary structure similar if not identical to that of its human counterpart. Also, the endogenous sugar transporter(s) present in Sf21 cells did not bind cytochalasin B, the potent transporter inhibitor. Therefore, the baculovirus/Spodoptera frugiperda (Sf) cell expression system could be very useful for production of large amounts of human glucose transporters, heterologously.

• International Journal of Industrial Entomology and Biomaterials
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• 제37권1호
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• pp.15-20
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• 2018

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (ERO1s) supply oxidizing equivalent to the active centers of PDI. We previously identified and characterized the ERO1 of Bombyx mori (bERO1) as a thioredoxin-like protein that shares primary sequence homology with other ERO1s. Here we compare the reactivation of inactivated rRNase and sRNase by bERO1, and show that bERO1 and bPDI cooperatively refold denatured RNase A. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

• 대한수의학회지
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• 제55권4호
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• pp.227-232
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• 2015

Akabane and bovine ephemeral fever (BEF) viruses cause vector-borne diseases. In this study, inactivated Akabane virus (AKAV)+Bovine ephemeral fever virus (BEFV) vaccines with or without recombinant vibrio flagellin (revibFlaB) protein were expressed in a baculovirus expression system to measure their safety and immunogenicity. Blood was collected from mice, guinea pigs, sows, and cattle that had been inoculated with the vaccine twice. Inactivated AKAV+BEFV vaccine induced high virus neutralizing antibody (VNA) titer against AKAV and BEFV in mice and guinea pigs. VNA titers against AKAV were higher in mice and guinea pigs immunized with the inactivated AKAV+BEFV vaccine than in animals inoculated with vaccine containing revibFlaB protein. Inactivated AKAV+BEFV vaccine elicited slightly higher VNA titers against AKAV and BEFV than the live AKAV and live BEFV vaccines in mice and guinea pigs. In addition, the inactivated AKAV+BEFV vaccine was safe, and induced high VNA titers, ranging from 1 : 64 to 1 : 512, against both AKAV and BEFV in sows and cattle. Moreover, there were no side effects observed in any treated animals. These results indicate that the inactivated AKAV+BEFV vaccine could be used in cattle with high immunogenicity and good safety.

• BMB Reports
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• 제37권6호
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• pp.735-740
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• 2004

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.964-967
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• 2008

Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.

• 한국잠사곤충학회지
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• 제37권1호
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• pp.46-51
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• 1995

국내 분리주의 BmNPV를 이용하여 유용 단백질을 생산할 수 있는 새로운 전이벡터의 제작과 외내 유전자의 발현에 대한 연구결과는 다음과 같다. 1. PCR 기법에 의하여 다각체 단백질 유전자의 ＋1 - －194에 해당하는 promoter 부위를 증폭하고 클로닝 하였다. 2. 다각체 단백질 유전子의 5' 및 3'의 leader 부위를 promoter 부위와 함께 단계적으로 클로닝하여 전이벡터를 완성하였다. 3. 완성된 전이벡터는 pBmKSK1으로 명명하였으며, 염기서열 결정을 통하여 다각체 단백질 유전자의 ＋2 부터 ＋597까지 제거되고 외내 유전자의 클로닝 sites로 EcoRI, KpnI과 SacI을 가짐을 확인하였다. 4. 외래 유전자로서 E. coli의 $\beta$-galactosidase 유전자를 pBmKSKl에 클로닝하고 누에 세포주에 전이시킨 후, X-gal 염색 및 SDS-PAGE에 의하여 pB- mKSKl이 제 기능을 수행함을 확인하였다.

• 한국응용곤충학회지
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• 제38권2호
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• pp.145-149
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• 1999

야생주 핵다각체병 바이러스의 낮은 병원성에 대해 살충제로서의 파밤나방 핵다각체병 바이러스(Spodoptera exigua nucleopolyhedrovirus: SeNPV)의 병원성 향상을 꾀함과 동시에 재조합 바이러스가 다각체 내에 매립됨으로써 살충제로의 적용시 야외에서의 안정성을 확보할 수 있는 p10 유전자의 프로모터를 이용한 새로운 발현벡터를 개발하기 위하여 국내분리주 SeNPV의 p10 유전자 구조를 분석하였다. 그 결과, SeNPV p10 유전자의 프로모터와 구조유전자 부위를 포함한 545염기서열을 결정하여 기존에 보고된 SeNPV p10 유전자(Zuidema et al., 1993)와 비교한 결과 구조유전자 부위에서는 100%의 상동성을 보였으나 5` 및 3` flanking region의 4개의 염기서열에서 차이를 보였다. Southern hybridization에 의하여 SeNPV전체 genomic DNA상에서 p10 유전자는 Sph I 2.4Kb와 Cla I 4.0Kb 단편내에 각각 존재함을 확인하였으며, p10 유전자를 포함하는 이들 단편을 각각 클로닝하여 제한효소 지도를 작성한다.