Generation of a Mammalian Gene Expression Vector Using Bovine Viral Diarrhea Virus

Bovine Vira1 Diarrhea Virus를 이용한 포유동물세포 발현벡터의 개발

  • 이영민 (충북대학교 의과대학 미생물학교실, 의학연구소)
  • Published : 2002.06.01

Abstract

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

최근 인간을 비롯한 다양한 생명체의 genome project연구결과 밝혀진 유전자들의 염기서열을 토대로, 생명체 구성성분의 실질적 인 역할을 하는 단배질의 기능을 밝히는 proteomics에 관한 연구의 필요성 이 대두되고 있다. 따라서, 이 연구는 post-genomics시대에 다양한 종류의 단백질 기능과 상호작용의 기초연구에 필수적 인 새로운 포유동물세포 유전자 발현벡터를 RNA 바이러스인 소설사성 바이러스(Bovine Viral Diarrhea Virus)의 infectious CDNA molecular clone을 이용하여 개발하였다. 먼저 BVDV의 infectious CDNA molecular clone (pNADLclns-)을 이용하여 puromycin 항생제에 저항성을 나타내는 puromycin acetyltransferase (pac) 유전자를 삽입하여 recombinant full-length infectious CDNA clone을 합성하였다. 합성된 recombinant CDNA clone을 주형으로 T7 RNA polymerase를 사용하여 in vitro transcribed full-length viral RNA를 합성하였다. 합성된 viral RNA의 자가복제 여부는 MDBK세포에 transfection시킨 후, $^{32}P$ 로 metabolically label함으로써 확인하였다. 또한, transfection된 세포에서의 바이러스 단백질 발현여부는 바이러스에 특이적으로 반응하는 anti-NS3 단클론항체를 사용하여 분석하였다. 또한, infectious CDNA clone을 응용하여 새로운 포유동물세포유전자 발현벡터의 개발을 위해서, 먼저 바이러스의 구조단백질이 바이러스의 자가복제에 필수적인 지를 평가하였다. 실험결과, 각각의 구조단백질 유전자를 deletion한 recombinant cDNA clone으로부터 합성된 viral RMA의 자가복제여부는pac유전자의 발현여부로 recombinant cDNA clone으로부터 합성된 recombinant viral RMA를 MDBK 세포에 transfection시킨 후, puromycin으로 selection함으로써 할 수 있었다. Deletion실험결과, 각각의 구조단백질 capsid및 E0, El, E2는 바이러스의 자가복제에 영향을 기치지 않음을 알 수 있었다. 이와 더불어, 바이러스의 모든 구조단백질을 함께deletion하였을 경우에도 자가복제에는 영향을 기치지 않는 것을 합성된 viral replicon을 이용한 실험에서 알 수 있었다. 이렇게 합성된 BVDV의 replicon을 사용하여 포유동물의 발현벡터로써 사용할수 있는 지의 여부를 분석하기 위해서 pac유전자 이외에 luciferase유전자를 사용하여 MDBK및 HeLa, BHK세포에서의 단백질 발현정도를 시간 별로 분석한 결과, BVDV의 replicon을 다양한 종류의 유전자 발현벡터로사용할 수 있음을 알 수 있었다. 그러므로, RNA바이러스의 하나인 BVDV의 viral replicon을 이용하여 다양한 종류의 포유동물 세포에 유전자 발현벡터로써 사용할 수 있음으로 post-genomics시대에 다양한 종류의 단백질 기능연구에 맡은 도움이 되리라 기대한다.

Keywords

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