CONCLUSIONS

To obtain TP-specific scFv, antibody selection system by a ribosome display was employed. Firstly a ribosome display technique was established using anti-DNA antibody, 3D8, as a model to select antibody and then, this system was applied to TP-specific antibody. TP-peptide-TP-specific selection and enrichment by ribosome display were shown. After four rounds of selection, the selected pool was cloned into an expression vector and each clone was screened by ELISA. Four TP-specific scFv clones were selected and their sequences were analyzed. These four selected scFvs have intact scFv sequence and specific binding activity to TP-peptide and HBV DNA polymerase protein.

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- 국문요약 -

R i b o s o m e d i s p l a y 를 이용한

B 형 간염바이러스 중합효소에 대한 s c F v 의 선별

아주대학교 대학원 이 명 신 (지도교수: 신 호 준)

목적: 기존에 보고된 eukaryotic translation system을 사용한 ribosome display 방법을 토대로 하여 항체 선별 방법을 확립하고, 이 확립된 방법을 이용하여 마우스 scFv 라이브러리로부터 B형 간염 바이러스 DNA 중합효소의 terminal protein영역에 특이적인 항체를 선별하고자 한다.

재료 및 방법: Ribosome display에 의한 항체 선별 방법을 확립하기 위하여 항-DNA 항체를 사용하였다. 항-항-DNA 항체, 3D8 scFv의 유전자를 시험관 내에서 전사-번역하였다. 번역된 3D8 scFv와 이를 코딩하는 mRNA는 리보솜에 연결되어 항체-리보솜-mRNA의 복합체를 형성하게 된다. 이 복합체를 특정항원과의 결합에 의해 선별하였고, 이 복합체로부터 분리된 mRNA를 역전사하여 PCR로 증폭하였다. 이 방법을 라이브러리로부터 항체 선별하는데 적용하기 위하여, B형 간염 바이러스 DNA 중합효소의 TP 영역의 57-80 아미노산과 일치하도록 합성된 TP 펩타이드를 마우스에 면역하고, 면역된 마우스의 비장을 분리하여 재조합

PCR방법으로 항체 라이브러리를 제작하였다. 제조된 항체 라이브러리를 이용하여 시험관내 합성에 의해 항체-리보솜-mRNA 복합체를 형성하였고, 이 복합체를 이용하여 TP 펩타이드에 대해 4회 선별하였다. 라이브러리 내의 TP 펩타이드에 대한 항체 증가를 방사선면역측정법으로 조사하였고, 4회 선별을 거친 라이브러리는 항체 발현을 위해 클로닝을 시행하였다. 클로닝 후, TP 펩타이드에 특이적인 항체 클론을 선별하였고, 선별된 클론은 염기서열을 분석하고 ELISA로 B형 간염 바이러스 DNA 중합효소에 대한 결합 능력을 확인하였다.

결과: Ribosome display를 이용한 항체 선별의 모델로써, 항-DNA 항체를 적용하였다. 항-DNA항체인 3D8의 유전자가 ribosome display에 의해 ssDNA에 대해 특이적으로 선별되는 것을 확인하였다. 이 실험을 통해 ribosome display에 의한 항체 선별방법을 확립하였다. 확립된 항체 선별 방법을 적용하여 면역된 마우스 라이브러리로부터 TP 펩타이드 특이 scFv를 4회 선별하였다. 선별을 통해 TP 펩타이드에 특이적인 라이브러리의 증가를 확인할 수 있었고, 선별된 scFv들은 TP 펩타이드와 B형 간염 바이러스 DNA 중합효소에 대한 결합력을 보였다.

결론: 진핵세포 translation에 의한 ribosome display 방법을 적용하여 항체 선별 방법을 확립하였다. 이 방법을 이용하여 항-TP scFv를 성공적으로 선별하였다.

핵심되는 말: 항체, B형 간염 바이러스, B형 간염 바이러스 DNA 중합효소, 라이브러리, Ribosome display, scFv, Terminal protein (TP)