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전형적인 PMWS (Post-weaning Multisystemic Wasting Syndrome, 이유후 전신소모성 증후군) 증세를 보이는 자돈의 임파절을 초대배양된 돼지 신장세포에 접종하여 porcine circovirus type 2 (PCV type 2)를 분리하였다. 분리한 virus를 확인하기 위하여 PCV type 2에 특이적인 primer를 사용한 중합효소 연쇄 반응법(Polymerase Chain Reaction, ...

전형적인 PMWS (Post-weaning Multisystemic Wasting Syndrome, 이유후 전신소모성 증후군) 증세를 보이는 자돈의 임파절을 초대배양된 돼지 신장세포에 접종하여 porcine circovirus type 2 (PCV type 2)를 분리하였다. 분리한 virus를 확인하기 위하여 PCV type 2에 특이적인 primer를 사용한 중합효소 연쇄 반응법(Polymerase Chain Reaction, PCR)을 이용하였다. PCV type 2의 target gene 에 반응하는 primer를 사용하여 ORF(Open Reading Frame) 1 및 2를 증폭하였다. 이 gene들을 cloning하여 baculovirus expression vector에 삽입하고, PCV의 재조합 단백질을 baculovirus expression system에서 발현하였다. 발현된 단백질은 PCV에 특이적인 polyclonal antibody를 이용하여 간접 형광 항체법 (indirect fluorescent assay, IFA)과 immunoblotting 방법을 통하여 증명하였다. ORF1은 baculovirus system에서 약 17kDa 및 31kDa 크기의 두개의 단백질을 발현하였다. ORF2의 재조합 단백질은 다른 분자량의 minor band 2개가 나타난 것을 제외하면 native virus 와 유사하였고 tunicamycin 처리시 최소한 2개의 glycosylation site를 가지고 있었다. ORF2의 재조합 단백질은 재조합 baculovirus 에 감염된 곤충 세포에서 virus-like particle을 형성하는 것을 확인하였다.

Abstract ▼ AI-Helper

Porcine circovirus (PCV) type 2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS (Post-weaning Multisystemic Wasting Syndrome). The presence of the virus was identified by PCR using primers specific to PCV type 2. The ORFs 1 and 2 were amplified by PCR usi...

Porcine circovirus (PCV) type 2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS (Post-weaning Multisystemic Wasting Syndrome). The presence of the virus was identified by PCR using primers specific to PCV type 2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immuno-blotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells.

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