• 한국응용곤충학회지
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• 제28권3호
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• pp.105-112
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• 1989

누에(Bombyx mori)와 흰불나방(Hyphantria cunea)으로 부터 분리된 핵다각체병바이러스(nuclear polyhedrosis virus: NPV)를 동정하기 위하여 전자현미경 관찰한 결과, BmNPV 다각체의 크기는 $3 \mu\textrm{m}$ 정도의 18면체로 외형이 균일하였으나, HcNPV는 1.5-$2 \mu\textrm{m}$ 정도이며 부정형이었다. Alkaline protease를 부활화시킨 후 SDS-PAGE한 다각체 단백질의 分子물은 BmNPV가 30 KD, HcNPV는 31 KD인 major band와 이들의 중합체(polymer)로 생각되는 57 KD, 112 KD의 minor band들이 관찰되었다. 또한 virion 단백질을 SDS-PAGE한 후 은염색한 결과, BmNPV는 분자량 9.6~112 KD인 47개의 band, HcNPV 경우는 분자량 9.4~l11 KD인 48개는 band가 관찰되었다. BmNPV와 HcNPV DNA의 제한효소 처이에 의한 전기영동 패턴을 관찰했으며, 각각의 genome 크기는 BmNPV가 약 116.4 Kb, HcNPV의 약 114.6 Kb였다.

• 한국응용곤충학회지
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• 제32권1호
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• pp.51-60
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• 1993

흰 불나방 핵 다각체바이러스(Hyphantria cunea nuclear Palyhedrosis virus: HcNPV) 다각체 단백질 유전자포함 절편의 탐색과 클로닝을 행하였다. Autographa cahfornica NPV EeoRI-I 절편 (약 7.3 kb), Bombyx mori NPV PstI-F 절편 (약 7 kb) 및 합성 oligonucleotide ( 3D-mer) 를 probe로 한 southern hybridization을 행하여 HcNPV PstI - L 절편 (5.3 kb)을 탐색하고, pUC18을 이용하여 E. coli에 형질전환시켜 클로닝하였다. 클로닝한 plasmid의 EeoRI, SaIl, Kpnl, HindIII, SacI 및 AvaI의 제한효소지도를 작성하고 pHeP-L(8.0 kb)이라 명명히였으며, 이를 다시 pHcP-Ll(4.7 kb), pHcP-L2(7.1 kb), pHcP-L3(5.3 kb), pHcP-L4(4.2 kb) 및 pHeP-L5(4.5 kb)로 subeloning 하였다.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.685-691
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• 1998

Baculovirus Hyphantrin. cunea nuclear polyhedrosis virus (HcNPV) insecticide containing the insecticidal protein (ICP) gene from Bacillus thuringiensis subsp. kurstaki HD1 was constructed using a lacZ-HcNPV system. The ICP ($\delta$-endotoxin) gene was placed under the control of the polyhedrin gene promoter of the HcNPV. A polyhedrin-negative virus was derived and named ICP-HcNPV insecticide. Then, the insertion of the ICP gene in the ICP-HcNPV genome was confirmed by Southern hybridization analysis. Polyacrylamide gel electrophoresis (PAGE) analysis of the Spodoptera frugiperda cell extracts infected with the ICP-HcNPV showed that the ICP was expressed in the insect cells as 130 kDa at 5 days post-infection. The ICP produced in the cells was present in aggregates. When extracts from the cells infected with the ICP-HcNPV were fed to 20 Bombyx mori larvae, the following mortality rate was seen; 8 larvae at 1 h, 10 larvae at 3 h, and 20 larvae at 12 h. These data indicate that the B. thuringiensis ICP gene was expressed by the baculovirus insecticide in insect cells and there was a high insecticidal activity. The biological activities of the recombinant virus ICP-HcNPV were assessed in conventional bioassay tests by feeding virus particles and ICP to the insect larvae. The initial baculovirus insecticide ICP-HcNPV was developed in our laboratory and the significance of the genetically engineered virus insecticides is discussed.

• International Journal of Industrial Entomology and Biomaterials
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• 제22권2호
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• pp.75-82
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• 2011

To elucidate the novelty of Hyphantria cunea nucleopolyhedrovirus (HcNPV) isolated in Korea, polyhedrin and inhibitor of apoptosis (iap) gene structures were determined and analyzed. The analysis of HcNPV polyhedrin showed a little difference with 97.6% at the nucleotide level but no difference at the amino acid level when compared with that of previously reported H. cunea NPV (HycuNPV). On the other hand, iap genes showed variable differences with 89.0-99.6% nucleotide and 90.0-99.6% amino acid sequence identities. Especially, the 5' and 3' non-coding flanking sequences of iap1 gene had lower degree of identity with those of HycuNPV. Although the phylogenetic analyses using polyhedrin and iap genes showed that HcNPV is closely related with HycuNPV, we could provide that HcNPV is a novel isolate having novel gene structures.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.355-362
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• 2000

The Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) gene in the pHLA-21 plasmid was inserted into a baculovirus (Hyphantria cunea nuclear polyhedrosis virus) expression vector (lacZ-HcNPV) to construct a recombinant virus gB-HcNPV expressing gB. Spodoptera frugiperda cells infected with this recombinant virus synthesized and processed gB of approximately 120 kDa, which cross-reacted with the monoclonal antibody to gB. The recombinant gB was identified on the membrane of the insect cells using an immunofluorescence assay. Antibodies to this recombinant raised in mice recognize the viral gB and neutralized the infectivity of the HSV-1 in vitro. These results show that the gB gene has the potential to be expressed in insect cells. They also demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the lacZ-HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• 한국응용곤충학회지
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• 제27권4호
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• pp.211-218
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• 1988

담배거세미나방(Spodoptera litura: SI), 누에(Bombyx mori: Bm) 및 흰불나방(Hyphantria cunea : Hc)으로부터 분리된 핵다각체병 바이러스(nuclear polyhedrosis virus : NPV)의 다각체의 단백질의 특성과 그에 대한 alkaline protease의 영향을 SDS-PAGE 및 혈정학적 방법으로 분석했다. Alkaline protease를 부활화시킨 후 다가체 단백질을 SDS-PAGE한 결과, BmNPV의 경우 30kD, SINPV와 HcNPV는 31kD의 단일 major band 및 이것들의 종합체(polymer)인 57kD와 66kD의 minor band들이 관찰되었다. Alkaline protease의 부활화를 성략한 SI NPV 다각체를 알칼리 용액으로 시간 차이를 두고 처리한 후 SDS-PAGE한 결고, 알칼리 처리시간이 경과함에 따라서 alkaline protease활성에 의해 다각체 단백질이 일정한 pattern으로 저분자화됨이 뚜렷하였다. SINPV 와 BmNPV 다각체 단백질의 항체를 제조하여 SINPV, BmNPV및 HcNPV 다각체 단백질간의 혈정학적 상동성을 이중형역광산법과 Western blot으로 비교한 결과는 3종 모두에서 공통 antigenic determinants의 존재가 인정되었으며 major 다각체 단백질의 종합체 형성과 alkaline protease에 의한 분해를 확인했다.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.541-547
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• 1999

Constructions of a transfer vector and a recombinant baculovirus using the thymidine kinase gene of the Herpes simplex virus type 1 strain F (HSV -1) were carried out. Newly cloned transfer vector, pHcgXIIIB, was constructed by insertion of the glycoprotein gX gene signal peptide sequence of Pseudorabies virus into the baculovirus vector pHcEV-IV. The gX sequence was inserted just downstream from the promoter for the polyhedrin gene of the Hyphantria cunea nuclear polyhedrosis virus (HcNPV). HSV-1 thymidine kinase(tk) gene (1.131 kb) was used as a candidate gene for transferring into the baculovirus expression system. The tk gene was inserted into a BamHI site downstream from the gX sequence-promoter for the polyhedrin gene in the pHcgXIIIB transfer vector and was transferred into the infectious lacZ-HcNPV expression vector. Recombinant virus was isolated and was named gX-TK-HcNPV. The recombinant virus produced a 45 kDa gX-TK fusion protein in Spodoptera frugiperda cells, which was confirmed by Western blot analysis. Microscopic examination of gX-TK-HcNPV-infected cells revealed normal multiplication. Fluorescent antibody staining indicated that the gX-TK fusion protein was present in the cytoplasm. These results indicated that the transfer vector successfully transferred the gX-tk gene into the baculovirus expression system.

• 한국어병학회지
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• 제8권1호
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• pp.37-46
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• 1995

H. cunea nuclear polyhedrosis virus (HcNPV) 의 polyhedrin 아미노산 및 polyhedrin gene 의 염기서열을 분석하였다. Polyhedrin 은 SDS-PAGE 상에서 3개의 polypeptide band 가 나타났고 주요 polypeptide 는 약 25 Kd 의 분자량을 갖고 있었다. 또한 polyhedrin 은 17 개의 다른 아미노산으로 구성되어 있었다. HcNPV DNA를 EcoRI 효소로 절단하여 $\alpha^{32}P$로 labelling 된 Autographa californica (AcNPV) polyhedrin gene cDNA 의 probe DNA를 이용하여 hybridization 한 결과 polyhedrin gene은 EcoRI 절편들중 H 절편에 양성반응을 나타냈다. 또한 polyhedrin gene 을 포함하고 있는 EcoRI-H 절편을 pUC8 벡터에 cloning한 다음 이를 hPE-H라고 이름하였다. HcNPV genome DNA 의 promoter 부위를 sequence한 결과 TATA box의 염기배열은 polyhedrin gene 전사 개시위치로부터 위쪽으로 -79 bp 의 5' flanking 부위에서 발견되었다. polyhedrin gene 내 CAAT box는 TATA box 측면 염기 배열에서 나타나지 않았고, 4개의 tandem repeat 5'-CTAATAT-3' 와 5'-TAAATAA-3'의 염기는 polyhedrin gene내 전이 개시 위치로 부터 위쪽으로 -141 과 -108 bp 또는 -83 bp 부위에 존재하였으며, 다른 하나는 전이 개시위치로 부터 아래쪽으로 -52 bp 부위에서 발견되었다. 그리고 polyhedrin gene 내 전이 개시위치로 부터 위쪽으로 -141 bp 부위는 다량의 AT (78%) 염기가 존재하였다. 또한 polyhedrin 의 개시 coding region 은 ATG 였고 종결 coding region은 TAA 였다.

• BMB Reports
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• 제33권6호
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• pp.483-492
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• 2000

The Herpes simplex virus type 1 (HSV-1) strain F glycoprotein H (gH) gene in the pHLB-4 plasmid was recombinated into a baculovirus expression vector (lacZ-HcNPV) to construct a recombinant virus GH-HcNPV expressing gH. The sequences of gH and its expression were analyzed. The gH gene was located in the 6.41 kb BglII fragment. The open reading frame (ORF) of the gH gene was 2,517 bp and codes 838 amino acid residues. Insect cells infected with this recombinant virus synthesized a high level of the matured and gX-gH fusion protein with approximately 112 kDa. The fusion gH protein was localized on the membrane of the insect cells as seen by using immunofluorescence assay and accumulated in the cultured media by the SDS-PAGE and immunoprecipitation assays. The amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. Antibodies raised in mice to this recombinant protein recognized viral gH and neutralized the infectivity of HSV-1 in vitro. These results demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV; accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.