목적 : 재조합 아데노 연관 바이러스 rAAVCMVp53를 생산하기 위해서 3가지 플라스미드 pAAVp53, pAAVLacZ 벡터 플라스미드, packaging 플라스미드, helper 플라스미드를 사용하여 cotransfection 방법을 통해 아데노바이러스 오염이 없는 순수 rAAVCMVp53을 생산하고자 하였다. 연구 방법 : 벡터 플라스미드 (pAAVp53, pAAVLacZ), AAV를 암호한 부분에 2개의 p5 promoter를 가지는 플라스미드 pXX2 그리고 복제할 수 있는 능력을 지닌 플라스미드 pXX6 이용하여 293 세포주에 Ca++-phospate 침전법을 통해 cotransfection을 시행하고 CsCl 밀도구배 초원심분리를 이용하여 재조합 아데노 연관 바이러스를 생산하였다. 생성과 적정확인은 PCR과 dot-blot을 통해 분석하였다. 결과 : PCR 법에 의해 p53과 LacZ 유전자를 가지는 재조합 아데노 연관 바이러스의 생성을 확인하였으며 LacZ 유전자를 포함하는 재조합 아데노 연관 바이러스를 자궁경부암 세포주 HeLa, HT3, HeLaS3, CaSki 등에 감염시킨 후 X-gal 염색을 통해 이입을 확인하였다. 결론 : 벡터플라스미드, packaging 플라스미드, helper 플라스미드를 이용하여 cotransfection을 통한 아데노바이러스의 오염이 없는 재조합 아데노 연관 바이러스를 생산할 수 있게 되었다. 향후 cotransfection의 자세한 연구를 통해 높은 titer의 순수한 재조합 아데노 연관 바이러스를 다량으로 생산할 수 있을 것으로 사료된다.

Objective : To eliminate the potential problem of adenovirus contamination during recombinant adeno- associated virus (rAAV) vector production, we investigated new rAAV production method by a triple transfection of vector plasmid, packaging plasmid, and adenovirus helper plasmid. Methods : This study was carried by triple transfection for the production of recombinant adeno- associated virus vector. This new production system was conducted with a plasmid construct which contained a mini-adenovirus genome capable of propagation of rAAV in the presence of adeno-assoceated viral rep and cap genes. To examine the helper functions of adenoviral plasmid on the production of rAAV vector, we carried out cotransfection with three plasmids, AAV vector, packaging construct, and adenovirus helper plasmids. The optimized plasmid quantity of transfection by calcium phosphate precipitation method was 25 µg of total plasmid DNA per 10 cm diameter plate of 293 cell. Results : We found that rAAV yields peaked at 48 hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/mL based on the quantification of viral DNA. Conclusion : We constructed recombinant AAVp53 without adenovirus contamination. We thought that we construct high titer rAAVp53 particle through HPLC column in the near future

Objective : To eliminate the potential problem of adenovirus contamination during recombinant adeno- associated virus (rAAV) vector production, we investigated new rAAV production method by a triple transfection of vector plasmid, packaging plasmid, and adenovirus helper plasmid. Methods : This study was carried by triple transfection for the production of recombinant adeno- associated virus vector. This new production system was conducted with a plasmid construct which contained a mini-adenovirus genome capable of propagation of rAAV in the presence of adeno-assoceated viral rep and cap genes. To examine the helper functions of adenoviral plasmid on the production of rAAV vector, we carried out cotransfection with three plasmids, AAV vector, packaging construct, and adenovirus helper plasmids. The optimized plasmid quantity of transfection by calcium phosphate precipitation method was 25 µg of total plasmid DNA per 10 cm diameter plate of 293 cell. Results : We found that rAAV yields peaked at 48 hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/mL based on the quantification of viral DNA. Conclusion : We constructed recombinant AAVp53 without adenovirus contamination. We thought that we construct high titer rAAVp53 particle through HPLC column in the near future

Adeno-associated virus, Cotransfection, Packaging plasmid, Helper plasmid, Cervical cancer