• 대한수의학회지
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• 제36권3호
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• pp.627-633
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• 1996

To analyze the genomic diversity of transmissible gastroenteritis virus (TGEV), the N-terminal half of the spike (S) glycoprotein gene and nonstructural protein gene (open reading frames 3 and 3-1) were amplified by reverse transcriptase reaction and polymerase chain reaction (RT-PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. In this study, TGEV Miller (M6) and Purdue (P115) strains were used as reference strains, and two vaccine strains (MSV and STC3) and four Korea isolates (P44, VRI-WP, VRI-41, and VRI-48) were analyzed. All TGEV strains were amplified with three TGEV primer pairs. Although there was some exception in RFLP analysis, this method differentiated TGEV strains into following groups : Miller group (M6 and MSV), Purdue group (PUS, STC3, P44, VRI-WP, VRI-41, and VRI-48). Using Sau3AI and SspI, VRI-48 was differentiated from the Miller and Purdue type viruses. The RT/PCR in conjuction with RFLP analysis was a rapid and valuable tool for differentiating several strains of TGEV. This study revealed the occurences of distinct difference in genome of TGEV strains.

• 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.

• 대한골관절종양학회지
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• 제3권1호
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• pp.9-17
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• 1997

Resistance to combination chemotherapy remains challenge in the treatment of osteosarcoma. One of the mechanisms of multiple drug resistance is an increased expression of the multidrug resistance gene(mdr1). Expression of the P-glycoprotein(mdr-1 gene product) was studied immunohistochemically and the mdr-1 gene by in situ hybridization in 33 osteosarcomas relating to various prognostic factors. Thirty cases out of 33 osteosarcomas(90.9%) showed positive cytoplasmic reactions with P-glycoprotein and nineteen instances(57.6%) were strong positive(2+). The older(>20 years) and female patients revealed more intense immunohistochemical reactions rather than those of the younger and male patients. Osteoblastic and chondroblastic osteosarcomas revealed more strong immunohistochemical reactions compared to fibroblastic types. There were no significant staining differences between the type of bony involvement, Broder's grade and the presence of necrosis. On follow-up, the mean survival rate was decreased in the strong positive group, however, this was not statistically significant. In situ hybridization for mdr-1 gene revealed positive signals in 22 cases out of 29 osteosarcomas(75.9%). Chemotherapy was done in 15 cases out of 28 patients(53.6%). The results of immunohistochemistry and in situ hybridization were not correlated with the protocols for chemotherapy. However, this result should be confirmed by a larger scale study about mdr1 mRNA expression.

• 한국어병학회지
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• 제33권2호
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• pp.103-109
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• 2020

In order to use nervous necrosis virus (NNV) virus-like particles (VLPs) as a delivery tool for heterologous antigens or plasmids, we attempted to produce red-spotted grouper nervous necrosis virus (RGNNV) VLPs displaying a partial region of viral hemorrhagic septicemia virus (VHSV) glycoprotein at the surface and VLPs that are harboring DNA vaccine plasmids within the VLP. A peptide encoding 105 amino acids of VHSV glycoprotein was genetically inserted in the loop region of NNV capsid gene, and VLPs expressing the partial part of VHSV glycoprotein were successfully produced. However, in the transmission electron microscope analysis, the shape and size of the partial VHSV glycoprotein-expressing NNV VLPs were irregular and variable, respectively, indicating that the normal assembly of capsid proteins was inhibited by the relatively long foreign peptide (105 aa) on the loop region. To encapsulate by simultaneous transformation with both NNV capsid gene expressing plasmids and DNA vaccine plasmids (having an eGFP expressing cassette under the CMV promoter), NNV VLPs containing plasmids were produced. The encapsulation of plasmids in the NNV VLPs was demonstrated by PCR and cells exposed to the VLPs encapsulating DNA vaccine plasmids showed fluorescence. These results suggest that the encapsulation of plasmids in NNV VLPs can be done with a simple one-step process, excluding the process of disassembly-reassembly of VLPs, and NNV VLPs can be used as a delivery tool for DNA vaccine vectors.

• 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.

• 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.

• 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 Plant Biology
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• 제36권2호
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• pp.127-132
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• 1993

Self-incompatibility in Brassica campestris is controlled by multi-allele system in a single genetic locus, the S locus, and it is elucidated that S-glycoproteins are S gene products. In this experiments, we examined the genetic mode(pollen tube behavior and segregation of S-glycoprotein), characteristic of S-glycoproteins and DNA constitution within nuclear genome on S gene family that unexplained by single locus model, and investigated the segregation pattern of S-glycoproteins in bred F1 generation. By diallel cross among the 15 plants within one family the existence of three types of homozygotes and three types of heterozygotes were observed, and segregation of S-allele could not explained by single locus model. From the results of IEF-immunoblot analysis for non-segregated individual plant, the segregation pattern of S specific bands was corresponded with results of diallel cross except with one case(SaSa genotype). The molecular weight of 6 different S-genotype varied in near by 50 kD, and each genotype expressed with 2 or 3 bands. Specific bands in SaSa, SbSb, ScSc has almost similar molecular weight between them. Southern analysis of genomic DNA probed with S-glycoprotein cDNA for 6 different genotypes revealed that there are clear difference in polymorphism, multiple bands of hybridization, when restriction enzymes of EcoR I were used. It could be assumed that there are several sequences related to the S-glycoprotein structural genes within their nuclear genome. Therefore, we suggested the possibilities that S-allele system could be controlled by multi-locus, that dominance-recessive interactions could be explained by modifier gene or supressor gene based on the results of abnormal segregation of S-glycoprotein in bred F1. The F2 analyses are progressing in now.

• 한국어병학회지
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• 제37권1호
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• pp.1-8
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• 2024

The advantages of replicon vectors of RNA viruses include a high ability to stimulate innate immunity and exponential amplification of target mRNA leading to high expression of foreign antigens. The present study aimed to construct a DNA-layered nervous necrosis virus (NNV) replicon vector system in which the capsid protein gene was replaced with a foreign antigen gene and to compare the efficiency of foreign antigen expression between the conventional DNA vaccine vector and the present replicon vector. We presented the first report of a nodavirus DNA replicon-based foreign antigen expression system. Instead of a two-vector system, we devised a one-vector system containing both an NNV RNA-dependent RNA polymerase cassette and a foreign antigen-expressing cassette. This single-vector approach circumvents the issue of low foreign protein expression associated with the low co-transfection efficiency of a two-vector system. Cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 (with the capsid gene ORF replaced with VHSV glycoprotein ORF) exhibited significantly higher transcription of the VHSV glycoprotein gene compared to cells transfected with either a vector without hammerhead ribozyme or a conventional DNA vaccine vector expressing the VHSV glycoprotein. Furthermore, the transcription level of the VHSV glycoprotein in cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 showed a significant increase over time. These results suggest that NNV genome-based DNA replicon vectors have the potential to induce stronger and longer expression of target antigens compared to conventional DNA vaccine vectors.

• 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.