• International Journal of Industrial Entomology and Biomaterials
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• 제1권1호
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• pp.53-58
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• 2000

A cDNA encoding the luciferase of firefly Luciola lateralis was cloned downstream from the polyhedrin gene promoter of Bombyx mori nucleopolyhedrovirus and expressed in B. mori cells (BmN-4). The coding soquence for luciferase was inserted into pBmKSK2 rectors) which was reconstructed from the polyhedrin-based transfer vector pBmKSKl by modifying cloning sites. Recombinant virus, BmK2-LUCDF, containing the luciferase gene was selected and purified in BmN-4 cells. The emission of luminescence by luciferase was only detected in BmK2-LUCDF-infected cell extracts. This result indicates that the cloned new luciferase gene of firefly L. lateralis can be expressed efficiently in baculovirus expression system and used as a useful reporter gene.

• 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.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권1호
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• pp.35-38
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• 2007

In this study, ORF 46 of Bombyx mod nucleopolyhedrovirus(Bm46) fused with EGFP was expressed in Bombyx mod cell lines, larvae and pupae by BmNPV Bacmid system. Bm46 and EGFP were cloned into donor plasmid pFastBacHTb, which was transformed to competent DH10B cells containing helper and BmNPV bacmid by site-specific transposition. Recombinant bacmid was used to transfected BmN-4 cells to produce the recombinant baculovirus vBm-Bm46-EGFP. Recombination virus was injected into silkworm larvae and pupae. The expression of the fusion protein was monitored by examining green fluorescence using a fluorescent microscope. Intense fluorescence in cells and silkworm was observed at 4 days post-infection, indicating the Bm46-EGFP fusion gene was expressed successfully.

• 한국잠사곤충학회지
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• 제38권1호
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• pp.53-56
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• 1996

곤충세포주로 널리 이용되고 있는 Sf 세포주와 Bm 세포주의 분자생물학적 표식자를 탐색하기 위하여, 총 세포 단백질의 SDS-PAGE와 genomic DNA를 RAPD(Random Amplification of Polymorphic DNA) 방법으로 분석하였다. 그 결과, 총 세포 단백질 및 genomic DNA, 패턴에서 두 세포주를 뚜렷하게 구별할 수 있는 밴드를 탐색하였으며, 이들은 유용한 분자 생물학적 표식자로 이용될 수 있을 것이다.

• 한국응용곤충학회지
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• 제32권4호
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• pp.407-413
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• 1993

숙주범위가 서로 다른 Autographa californica NPV(AcNPV)와 Bombyx mori NPV(BmNPV)를 Spodoptera frugiperda(Sf9) 또는 Bombyx mori(BmN-4)의 세포에 동시감염(coinfection)시킨 후, 숙주범위가 확장된 재조합 바이러스를 Sf9세포에서 10종 BmN-4세포에서 2종씩 플라크 순화하여 선발하였다. 각 재조합 바이러스 DNA의 제한효소 분석결과는 한번 이상의 재조합이 일어났음을 보여주었다. 재조합 바이러스 RecB-8의 전자현미경 관찰결과는 다각체의 모양이 모바이러스인 AcNPV나 BmNPV와는 전혀 다른 정사면체 모양이었으며 또한, 모바이러스와는 달리 virion이 다각체에 거의 매립되어 있지 않은 특징을 보였다.

• 한국잠사곤충학회지
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• 제40권1호
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• pp.60-62
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• 1998

To understand expression of some early and late genes of Bombyx mori nuclear polyhedrosis virus (BmNPV) in the B. mori-derived BmN cell line, the transcripts were analyzed by polymerase chain reaction with synthetic primers. After infection, the transcript of early genes, which include p35, IE1 and helicase p143, was immediately detected in the infected cells. In addition, the transcript of late genes, which include p10 and polyhedrin, was also detected in just-infected cells. In conclusion, our results revealed that transcripts of early and late genes of BmNPV are immediately expressed from the cells after infection.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.72-76
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• 1996

To investigate the expression efficiency of new transfer vector of Bombyx mori nuclear polyhedrosis virus (BmNPV), Escherichia coli lacZ gene was inserted into new transfer vector pBmKSK1, under the control of polyhedrin promoter and expressed in BmN-4 cells and larvae of silkworm, Bombyx mori. The recombinant virus containing lacZ gene was isolated from BmN-4 cells coinfected with transfer vectro pBmKSK1-LacZ and wild type BmNPV genome, and analysed by Southern blotting. The expression of ${\beta}$-galactosidase was characterized by SDS-PAGE, Western blotting and ${\beta}$-galactosidase activity assay. The results showed that the level of expression in silkworm larvae was higher than that of BmN-4 cells.

• 한국잠사곤충학회지
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• 제37권1호
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• pp.52-56
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• 1995

Baculovirus 발현계에서 BmNPV의 증식효율을 향상시키기 위하여, 누에 유충 체액의 BmN-4 세포주에서 BmNPV 증식에 미치는 영향을 조사하였다. 5령 3일째의 누에 유충으로 부터 체액을 추출하여 BmN-4 배양액에 첨가한 결과, 열처리하지 않은 누에 체액의 첨가는 세포의 응집과 소형화 현상으로 인해 세포의 증식을 저해한 반면, $65^{\circ}C$에서 30분간 열처리한 체액 10%와 FBS 3%를 세포배양액에 첨가한 것이 바이러스 감염에 의해 방출된 다각체의 수가 가장 많은 것으로 나타나 바이러스 증식에 가장 효과적이었다. 또한 plaque assay 결과, 체액의 첨가에 의한 바이러스 증식효율의 증가는 세포의 증식에 의한 것이라기보다 바이러스 감염성의 증가에 기인하는 것으로 보여진다.

• 한국잠사곤충학회지
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• 제33권1호
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• pp.21-26
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• 1991

Bm 배양세포주(BmN4)에서 BmNPV의 다각체 단백질 합성과 DNA복제에 대한 실험결과는 다음과 같다. 1. BmNPV는 insect Grace's medium에서 배양되고 있는 BmN4 세포에서 NOV나 DNA로 감염시킨 경우 모두 잘 증식되었으며, 도립현미경 관찰 결과 접종 후 48시간이 경과하면서 다각체가 형성되기 시작하였다. 2. 또한 전자현미경으로 핵내 형성된 다각체와 협성중인 nucleocapsid 다발을 관찰하였으며, 바이러스 입자는 SNPV로 존재하였다. 3. Western blot분속에 의한 다각체 단백질의 합성은 접종 후 18시간부터 관찰되었으며, 다각체 단백질의 분자량은 30kd이었다. 4. Wild type BmNPV DNA와 Bm 세포(BmN4)에서 NOV 및 DNA 감염에 의해 형성된 바이러스 DNA간의 제한효소 패턴은 차이가 없었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권2호
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• pp.131-135
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• 2007

Open reading frame 4 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm4, is a gene whose function is completely unknown. With the recently developed BmNPV bacmid and a modified pFastBac1 whose polyhedrin promoter was replaced with ie1 promoter, a recombinant bacmid expressing Bm4-EGFP fusion protein under the control of ie1 promoter in BmN cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced efficiently with other promoters to direct the expression of foreign gene in BmN cells by using Bac-to-Bac/BmNPV baculovirus expression system but also laid the foundation for rescue experiment of Bm4 deletion mutant due to the ability of ie1 promoter to direct gene expression throughout the infection cycle.