• 한국가축번식학회지
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• 제25권2호
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• pp.171-180
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• 2001

형질전환 가금의 생산에 있어서 retrovirus vector를 이용하는 방법은 다양한 종류의 표적세포에 대하여 retrovirus 고유의 감염성에 의한 외래 유전자의 전이가 용이하고, 전이된 유전자가 진정염색질 영역 내로 선택적으로 도입될 수 있으며 유전적으로 안정성을 나타내므로 매우 효과적인 방법이다. 그러나 가금에서는 초기 배발달에 의한 급격한 세포의 수적 증가로 인해 고감염성의 virus의 획득이 요구되므로, 이를 위하여 virus stock의 농축에 있어 보다 안정적이고 pantropic인 vesicular stomatitis virus (VSV G) glycoprotein를 envelope로 가지는 pseudotyped retrovirus vector system을 이용하였으며, marker gene으로 eGFP gene이 발현되는 retrovirus를 생산하였다. 이 virus를 이용하여 여러 가지 표적세포와 primary culture한 CEF세포를 감염시켜 GFP의 발현을 확인하였으며, 농축한 virus stock은 stage X의 계란을 선택하여 windowed egg를 제작한 후 배하층에 주입하였다. 형질전환 닭은 정상 발생한 닭에 비하여 저조한 발생율을 보였으나 PCR을 이용하여 외래 유전자의 도입을 확인한 결과 100%인 것으로 나타났다. 또한 한 개체 내에서 유전자의 도입이 폐, 간, 정소, 소장 등의 여러 장기에서 확인되었다.

• BMB Reports
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• 제34권4호
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• pp.371-378
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• 2001

The Glycoprotein D (gD) gene of the HSV-1 strain F was cloned, sequenced, recombinated into the HcNPV (Hyphantria cunea nuclear polyhedrosis virus) expression vector and expressed in insect cells. The gD gene was located in the 6.43 kb BamHI fragment of the strainF. The open reading frame (ORF) of the gD gene was 1,185 by and codes 394 amino acid residues. Recombinant baculoviruses, GD-HcNPVs, expressing the gD protein were constructed. Spodoptera frugiperda cells, infected with the recombinant virus, synthesized a matured gX-gD fusion protein with an approximate molecular weight of 54 kDa and secreted the gD proteins into the culture media by an immunoprecipitation assay The fusion gD protein was localized on the membrane of the insect cells, seen by using an immunofluorescence assay The deduced 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. These results indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins.

• 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.

• The Plant Pathology Journal
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• 제15권6호
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• pp.313-318
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• 1999

Cloning of the 5'untranslated region (5' UTR) and Nterminus of the glycoprotein precursor (G2G1) open reading frame of tomato spotted wilt virus has been problematic, possibly because of the toxicity of a signal peptide at the beginning of th G2G1 protein precursor. The toxicity of the signal peptide to bacterial growth and the reason for the expression of the peptide gene in Escherichia coli were investigated by cloning the 5' UTR and the signal peptide sequence separately. Cells transformed with the plasmid containing both the first 30 amino acids of the glycoprotein and the 5' UTR showed a severe growth inhibition whereas transformants harboring either the plasmid with the signal sequence or the 5'UTR alone did not show any ingibition. An E. coli promoter-like sequence was found in the 5'UTR and tis promoter acivity was confirmed with a promoter-less GUS gene cloned downstream of the 5'UTR. In the cloning of the Tomato spotted wilt virus (TSWV) glycoprotein G2G1 open reading frame all the recovered plasmids contained stop codons in the signal sequence region. However, clones containing no stop codon were recovered when the signal sequence and the 5'UTR were cloned separately.

• 한국어병학회지
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• 제23권1호
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• pp.1-8
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• 2010

무지개 송어 발안란의 이동 이력과 IHNV G gene 염기서열의 계통학적 분석에 의해 IHNV의 국내 이동에 대해서 처음으로 조사되었다. IHNV RtWanju09 분리주는 IHNV RtPy91과 RtJe00의 JRt Shizuoka lineage에 속하는 것으로 나타났으며, 강원도에서 분리된 IHNV RtPy91과 RtWanju09 사이의 G gene의 유전적 차이는 1.77%였고, 약 20년간 우리나라의 JRt Shizuoka lineage에 속하는 한국 분리주들은 최대 3.03%의 유전적 차이를 나타내 계속적으로 바이러스가 진화하는 것을 알 수 있었다. 또한 전라북도 지역에서 분리된 IHNV RtWanju09 분리주는 IHNV가 오염된 강원도 지역산 발안란이 전라북도에 전파되었을 것으로 사료된다.

• 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.

• 대한바이러스학회지
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• 제29권2호
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• pp.99-105
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• 1999

BamHI restriction pattern and genomic library of Herpes simplex virus type 2 (HSV-2) strain G were constructed, and locations of the glycoproteins gB and gD, and tk genes on the fragments were detected by Southern blot analysis. HSV-2 genomic DNAs were cleaved into twenty-seven fragments by BamHI enzyme in the range of 0.72 to 15.08 (total 150.44 kb), which were cloned into the BamHI site of pBluescript SK(+) to construct genome library of the HSV-2. The library was named by the order of the fragment size from smallest one to largest one. HSV-2 glycoprotein gD gene was located in pHLA2-21 and pHLA2-22 recombinant plasmids, gB gene in pHLA2-24 plasmid, and tk gene in pHLA2-11 clone by Southern blot analysis.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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• pp.163-169
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• 2001

Despite the high potency of the retrovirus vector system in gene transfer, one of the main drawbacks of has been difficulty in preparing highly concentrated virus stock. Numerous efforts to boost the virus titer have ended in unsatisfactory results mainly due to fragile property of retrovirus envelope protein. In this study, to overcome this problem, we constructed our own retrovirus vector system producing vector viruses encapsulated with VSV-G (vesicular stomatitis virus G glycoprotein). Concentration process of the virus stock by ultracentrifuge did not sacrifice the virus infectivity, resulting in more than 108 to 109 CFU (colony forming unit) per ml on most of the target cell lines tested. Application of this high-titer retrovirus vector system was tested on chicken embryos. Injection of virus stock beneath the blastoderms of pre-incubated fertilized eggs resulted in chick embryos expressing E. coli LacZ gene with 100% efficiency. Therefore, our results suggest that it is possible to transfer the foreign gene into chicken embryo using our high-titer retrovirus vector.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.193-199
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• 2010

We report here the production of transgenic chickens that can regulate human erythropoietin (hEPO) gene expression. The glycoprotein hormone hEPO is an essential for viability and growth of the erythrocytic progenitors. Retrovirus vector system used in this study has two features including tetracycline-controllable promoter and woodchuck hepatitis virus posttranscriptional regulator element (WPRE). The former is for to reduce the possibility of physiological disturbance due to constitutional and unregulated expression of hEPO gene in the transgenic chicken. The latter is for maximum expression of the foreign gene when we turn-on the gene expression. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 325 injected eggs, 28 chicks hatched after 21 days of incubation and 16 hatched chicks were found to express the hEPO gene delivered by the vector. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. The recombinant hEPO in transgenic chicken serum had N- and O-linked carbohydrate simillar to that produced from in vitro cultured cells transformed with hEPO gene.