Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Expression, Purification and Antiserum Production of the Avian Influenza H9N2 Virus HA and NA Proteins

Avian Influenza H9N2 Virus의 HA와 NA 단백질 발현, 정제 및 항혈청 생산

  • Lee, Hyun-Ji (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Song, Byung-Hak (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kim, Jeong-Min (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Yun, Sang-Im (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kim, Jin-Kyoung (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kang, Young-Sik (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Koo, Yong-Bum (School of Biotechnology and Biomedical Science, Inje University) ;
  • Jeon, Ik-Soo (Research Planning Team, National Institute of Animal Science) ;
  • Byun, Sung-June (Animal Biotechnology Division, National Institute of Animal Science) ;
  • Lee, Youn-Jeong (National Veterinary Research and Quarantine Service) ;
  • Kwon, Jun-Hun (National Veterinary Research and Quarantine Service) ;
  • Park, Jong-Hyeon (National Veterinary Research and Quarantine Service) ;
  • Joo, Yi-Seok (National Veterinary Research and Quarantine Service) ;
  • Lee, Young-Min (Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University)
  • 이현지 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 송병학 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 김정민 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 윤상임 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 김진경 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 강영식 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 구용범 (인제대학교 의생명공학대학 생명공학부) ;
  • 전익수 (농촌진흥청 축산과학원 축산기획조정과) ;
  • 변승준 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 이윤정 (국립수의과학검역원) ;
  • 권준헌 (국립수의과학검역원) ;
  • 박종현 (국립수의과학검역원) ;
  • 주이석 (국립수의과학검역원) ;
  • 이영민 (충북대학교 의과대학 미생물학교실)
  • Published : 2008.09.30
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Abstract

Avian influenza virus (AIV) is recognized as key to the emergence of pandemic influenza for humans; there are growing concerns that AIV H9N2 may become more efficient to transmit to humans in the near future, since the infection of poultry with AIV H9N2 has been common in recent years. In this study, we aimed to produce antisera recognizing the HA and NA proteins of AIV H9N2. Initially, coding sequences corresponding to the N-terminal regions of the HA and NA proteins of the Korean AIV H9N2 (A/Ck/Kr/MS96/96) isolated from a domestic chicken were amplified from the genomic RNA. Following cloning of the amplified cDNA fragments into pGEX4T-1 vector, two GST-fusion proteins (GST-HAln and GST-NAn) were expressed in E. coli BL21 and purified with glutathione sepharose columns; the recombinant GST-HAln and GST-NAn proteins were both used as immunogens in rabbits. The antigenicity of the rabbit antisera was analyzed by immunoblotting of the cell lysates prepared from AIV H9N2-infected MDCK cells. Overall, the recombinant HAln and NAn proteins fused to the C-terminus of GST and the rabbit antisera raised against the corresponding recombinant proteins would provide a valuable reagent for AIV diagnosis and basic research.

조류 독감바이러스(avian influenza virus, AIV)는 사람에게서 발생하는 인플루엔자 대유행에 중요한 역할을 한다. 특히 최근 AIV H9N2형에 의한 가금류 감염이 빈번히 나타나고 있어 인체 감염이 상당히 우려되는 실정이다. 본 연구에서는 최종적으로 AIV의 HA와 NA 단백질에 특이적으로 반응하는 항혈청을 생산하고자 하였다. 먼저 감염된 닭에서 분리된 AIV H9N2 한국분리주 A/Ck/Kr/MS96/96의 게놈RNA로부터 RT-PCR 방법으로 HA와 NA 단백질 N-말단부위에 해당하는 염기서열을 증폭하였다. 이렇게 증폭된 DNA단편은 E. coli 발현벡터 pGEX4T-1에 삽입한 후, BL21 세포에서 각각의 GST fusion protein (GST-HAln와 GST-NAn) 형태로 발현하였다. GST-HAln와 GST-NAn은 모두 glutathione sepharose column을 사용하여 분리 및 정제하였으며, 정제된 단배질을 항원으로 사용하여 토끼 항혈청을 생산하였다. 생산된 항혈청의 항원특이성은 AIV H9N2 한국분리주 A/Ck/Kr/MS96/96로 감염된 MDCK 세포의 cell extract를 사용하여 immunoblotting을 수행함으로써 확인하였다. 본 실험결과AIV H9N2의 HA와 NA단백질 N-말단부위에 해당하는 재조합GST fusion protein과, 이들 각각의 단백질에 특이적으로 반응하는 항혈청은 앞으로AIV 감염의 진단 뿐만 아니라, AIV에 대한 기초연구에 중요한 재료로 사용될 것으로 기대한다.

Keywords

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