• Journal of Microbiology and Biotechnology
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• 제12권4호
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• pp.563-568
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• 2002

Stably transformed Drosophila melanogaster 52 cells producing recombinant VP7 were obtained, and recombinant VP7 expression was confirmed by Western blot analysis. The molecular weight of recombinant VP7 expressed in 52 cells was approximately 35.5 kDa, and 75% of the total VP7 produced was present in the medium. Recombinant VP7 contained N-linked glycosylated oligosaccharides. Aprotinin, leupeptin, and polyvinylpyrrolidone did not have any noticeable effect on recombinant VP7 production; however, DMSO and sodium butyrate increased its production by 120% and 60%, respectively.

• 한국어병학회지
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• 제28권2호
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• pp.93-98
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• 2015

Full-length recombinant VP2 and VP3 proteins of aquabirnavirus isolated from olive flounder were expressed successfully in E. coli expression system. After rats were immunized with these proteins, antisera were used for in vitro and in vivo neutralization test. In in vitro test, VP2 antibody titers were higher than that of VP3. In in vivo assays, fish challenged with aquabirnavirus neutralized with VP2 antibody survived longer than other fish.

• 대한수의학회지
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• 제43권3호
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• pp.449-456
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• 2003

The VP2 gene of aquatic birnavirus, Korean isolate (GC-1) was cloned and expressed using the baculovirus expression system. The VP2 gene and VP2 partial gene, which contained a neutralizing epitope, were constructed for recombinant transfer vectors, for baculovirus expression. The expressed recombinant proteins were confirmed by indirect immuno fluorescence antibody (IFA), SDS-PAGE and Western blot. The level of expression was checked at regular time using IFA and Western blot. To measure the neutralizing activity of recombinant proteins against GC-1 strain, the antisera against recombinant proteins were produced by using guinea pigs. The result showed that the antisera neutralized the GC-1 strain. However, the neutralizing titer was higher in antisera against the VP2 gene expressed recombinant protein than that of VP2 partial gene recombinant protein.

• 대한수의학회지
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• 제61권2호
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• pp.19.1-19.7
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• 2021

Feline panleukopenia virus (FPV) causes leukopenia and severe hemorrhagic diarrhea, killing 50% of naturally infected cats. Although intact FPV can serve as an antigen in the hemagglutination inhibition (HI) test, an accidental laboratory-mediated infection is concern. A non-infectious diagnostic reagent is required for the HI test. Here, we expressed the viral protein 2 (VP2) gene of the FPV strain currently prevalent in South Korea in a baculovirus expression system; VP2 protein was identified by an indirect immunofluorescence assay, electron microscopy (EM), Western blotting (WB), and a hemagglutination assay (HA). EM showed that the recombinant VP2 protein self-assembled to form virus-like particles. WB revealed that the recombinant VP2 was 65 kDa in size. The HA activity of the recombinant VP2 protein was very high at 1:2¹⁵. A total of 143 cat serum samples were tested using FPV (HI-FPV test) and the recombinant VP2 protein (HI-VP2 test) as HI antigens. The sensitivity, specificity, and accuracy of the HI-VP2 test were 99.3%, 88.9%, and 99.3%, respectively, compared to the HI-FPV test. The HI-VP2 and HI-FPV results correlated significantly (r = 0.978). Thus, recombinant VP2 can substitute for intact FPV as the serological diagnostic reagent of the HI test for FPV.

• 생명과학회지
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• 제20권8호
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• pp.1181-1185
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• 2010

본 연구는 WSSV의 주요 구조단백질인 VP19와 VP28을 모두 포함하는 VP19+28 fusion protein을 제조하여, Litopenaeus vannamei에서 WSSV에 대한 백신으로서의 효능을 평가하고자 수행하였다. VP19와 VP28 유전자를 fusion하여 제작한 VP19+28 유전자를 pET-28a(+) vector에 삽입하고 단일단백질로서 제작된 VP19+28 유전자를 E. coli BL21 (DE3)에서 발현시켰다. 백신실험을 위해 새우에게 2주 동안 실험용 사료를 급이하였으며, 그 후 바이러스액($1{\times}10^2$배로 희석한 WSSV)을 이용하여 새우에게 주사 감염에 의해 in vivo 공격실험(challenge test)을 수행하였다. 실험결과, vaccination을 하지 않은 새우들은 감염 후 11일째에 100%의 누적폐사율을 보였으며, host control로써 E. coli BL21을 사용하여 vaccination한 새우들은 감염 후 17일째에 100%의 누적폐사율을 보였다. rVP19, rVP28, rVP19+28을 이용하여 vaccination한 새우들의 경우 감염 후 21일째에 각각 66.7%, 41.7%, 41.7%의 누적폐사율을 보였다. 이상의 결과를 통해 rVP28과 rVP19+28이 WSSV에 대해 높은 백신효능을 가짐을 확인하였다. 또한 감염 후 21일째에 fusion protein rVP19+28과 rVP28의 누적폐사율은 동일하였지만 공격실험기간 동안 폐사율이 rVP19+28을 투여 한 실험군이 낮게 나타나는 것을 보아 WSSV에 대한 새우의 방어효능은 rVP19+28이 더 높음을 나타내는 것이다.

• Journal of Microbiology and Biotechnology
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• 제21권2호
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• pp.170-175
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• 2011

This study was carried out for the molecular level identification of recombinant protein vaccine efficacy, by oral feeding against white spot syndrome virus infection, with the comparison of viral mRNA transcriptional levels in shrimp cells. For the determination of WSSV dilution ratio for the vaccination experiment by oral feeding, in vivo virus titration was carried out using different virus dilutions of virus stock ($1{\times}10^2$, $2{\times}10^2$, and $1{\times}10^3$). Among the dilution ratios, $2{\times}10^2$ diluted WSSV stock was chosen as the optimal condition because this dilution showed 90% mortality at 10 days after virus injection. Recombinant viral proteins, rVP19 and rVP28, produced as protein vaccines were delivered in shrimps by oral feeding. The cumulative mortalities of the shrimps vaccinated with rVP19 and rVP28 at 21 days after the challenge with WSSV were 66.7% and 41.7%, respectively. This indicates that rVP28 showed a better protective effect against WSSV in shrimp than rVP19. Through the comparison of mRNA transcriptional levels of viral genes from collected shrimp organ samples, it was confirmed that viral gene transcriptions of vaccinated shrimps were delayed for 4~10 days compared with those of unvaccinated shrimps. Protection from WSSV infection in shrimp by the vaccination with recombinant viral proteins could be accomplished by the prevention of entry of WSSV due to the shrimp immune system activated by recombinant protein vaccines.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1960-1965
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• 2015

Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 μl and of the supernatant 4,096 units/50 μl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.

• 대한바이러스학회지
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• 제27권2호
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• pp.239-255
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• 1997

전염성 췌장괴저바이러스 (Infectious pancreatic necrosis virus) DRT 주의 VP1유전자를 대 장균 발현운반체와 Baculovirus에 삽입하여 대장균과 진핵세로에서 VP1단백질의 발현을 연구하였다. 재조합체 pMal-pol 클론 [7]에서 2.7 Kb 단편인 VP1 유전자를 제한효소 XbaI으로 절단하여 Baculovirus 운반체인 pBacPAK9에 클로닝하여 pBacVP1이라 명명하였다. 이 pBacVP1에 클로닝된 VP1유전자를 제한효소 SacI과 PstI으로 절단하여 대장균 발현 운반체인 pQE-30에 클로닝하여 pQEVP1이라 명명하였다. 또한 VP1 단백질의 C-말단에 6개의 히스티딘 $6{\times}His$이 붙어 있는 단백질을 만들기 위하여, pQEVP1 클론의 His부위를 EcoRI으로 절단하고, 또한 pBacVP1을 EcoRI으로 절단하여 생긴 부위에His-EcoRI DNA 단편을 교체시켜 재클로닝하여 pBacHis-VP1을 만들었다. pBacHis-VP1 DNA와 Bsu36I로 처리된 LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV)를 함께 lipofectin을 이용하여 곤충세포 (Spodoptera frugiperda cell)에 동시 감염을 시켜서 재조합 바이러스를 선발하여, VP1-HcNPV-1이라 명명하였다. pQEVP1 클론은 6개의 히스티딘 단편이 부착된 VP1단백질을 Ni-NTA resin 크로마토그래피법으로 정제하여 SDS-PAGE와 Western blot으로 확인하였고, 단백질의 활성과 구조에 영향을 주지 않는 6개의 히스티딘 단편 ($6\;{\times}\;His$)이 부착된 94 kDa의 VP1단백질을 정제할 수 있었다. 또한 재조합 바이러스에 감염된 곤충세포에서 VP1 단백질이 발현된 것을 전기영동과 Western blot으로 검색을 한 결과 95 kDa VP1 단백질이 발현이 되었음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.369-377
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• 1998

Rotavirus is a major cause of severe gastroenteritis in young children and animals throughout the world. The VP7 of rotavirus is thought to induce the synthesis of neutralizing antibodies and to be responsible for determining viral serotypes. The cDNA coding for the VP7 capsid protein of human rotavirus, obtained from Korean patients (HRV-Y14), was cloned and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequences between VP7 of Y14 and that of other foreign isolates showed $92.7~95.2\%$ homology to G1 serotypes (RV-4, KU, K8, WA), $74.2\%$ homolgy to G2 serotype HU-5, $76.4\%$ homology to G3 serotype SA-11, and $77.6\%$ homology to G4 serotype A01321. These data suggest that HRV-Y14 can be classified as a G1 serotype. cDNA coding for VP7 of HRV-YI4 was subcloned into the baculovirus vector and the VP7 glycoprotein was expressed in insect cells. The expressed proteins in Sf9 cell extract and tissue culture fluid were separated on SDS-PAGE, and Western blot analysis with monoclonal antibody raised against the synthetic peptide containing 21 amino acids within the VP7 conserved region was performed. The molecular weight of recombinant VP7 was estimated to be 36 kDa which is about the same size as the native VP7. Addition of tunicamycin in the culture media caused a reduction of the molecular weight of the recombinant VP7 indicating that the expressed protein was glycosylated.

• Biomolecules & Therapeutics
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• 제20권3호
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• pp.320-325
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• 2012

Rotavirus and hepatitis A virus (HAV) spread by the fecal-oral route and infections are important in public health, especially in developing countries. Here, two antigenic epitopes of the HAV polyprotein, domain 2 (D2) and domain 3 (D3), were recombined with rotavirus VP7, generating D2/VP7 and D3/VP7, cloned in a baculovirus expression system, and expressed in Spodoptera frugiperda 9 (Sf9) insect cells. All were highly expressed, with peak expression 2 days post-infection. Western blotting and ELISA revealed that two chimeric proteins were antigenic, but only D2/VP7 was immunogenic and elicited neutralizing antibody responses against rotavirus and HAV by neutralization assay, implicating D2/VP7 as a multivalent subunit-vaccine Candidate for preventing both rotavirus and HAV infections.