베큘로바이러스 감염 누에 (Bombyx mori) 유충에서 돼지 콜레라 바이러스 구조 당단백질 E2의 생산 : Production of Structural Glycoprotein E2 of Classical Swine Fever Virus in Baculovirus-infected Silkworm Bombyx mori Larvae

국문 초록 (Abstract)

돼지 콜레라 바이러스 (CSFV)의 구조 당단백질 E2 (gE2)는 돼지의 면역 반응을 방어하는 기능을 가진다. 본 연구에서 누에 (Bombyx mori) 유충을 베큘로바이러스에 감염시켜 CSFV gE2를 생산하였다. 172개의 아미노산으로 구성된 CSFV gE2ΔC 유전자를 다각체 단백질과 fusion시켜 재조합 바이러스 BmNPV-gE2ΔC를 제작하였다. BmNPV-gE2ΔC를 감염시킨 누에 유충에서 생산된 재조합 다각체 단백질은 다각체 항체와 CSFV E2 항체를 이용하여 약 50 kDa의 분자량으로 확인되었다. 재조합 다각체를 전자현미경과 공초점 현미경에서 관찰한 결과, 형태학상 정상적인 형태를 유지하며 CSFV gE2ΔC를 가지는 것을 증명하였다. 재조합 다각체 또는 유화시킨 수용성 fusion 단백질을 이용하여 쥐로부터 면역성을 유도한 결과, 재조합 다각체 단백질은 CSFV gE2ΔC에 대한 면역성을 가지는 것으로 나타났다. 이러한 결과는 CSFV gE2ΔC의 재조합 다각체는 granule 항체가 잠재적으로 백신으로서의 가능성을 나타낸다.

다국어 초록 (Multilingual Abstract)

Classical swine fever virus (CSFV) envelope glycoprotein E2 (gE2) is responsible for inducing a protective immune response in swine. Here we report the production of CSFV gE2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) expressing polyhedrin fused with a CSFV gE2ΔC gene encoding the N-terminal 172 amino acids of CSFV E2. The recombinant polyhedra expressed in BmNPV-gE2ΔC-infected silkworm larvae produced an approximately 50 kDa fusion protein that was detected by both anti-polyhedrin and anti-CSFV E2 antibodies. Both electron and confocal microscopy demonstrated that the recombinant polyhedra are morphologically normal and contained CSFV gE2ΔC. Mice immunized with recombinant polyhedra alone or an emulsified soluble form of the fusion protein elicited CSFV E2 antibodies, indicating that the recombinant polyhedra carrying CSFV gE2ΔC were immunogenic. These results demonstrate that the recombinant polyhedra carrying CSFV gE2ΔC as a granule antigen can be used as a potential vaccine against CSFV

목차 (Table of Contents)

• Ⅰ. INTRODUCTION.................................................................................1
• Ⅱ. MATERIALS AND METHODS.........................................................3
• 1. Insects.................................................................................................................3
• 2. Construction of recombinant baculovirus................................................3
• 3. Production of recombinant polyhedra.......................................................3
• 4. Antibody preparation and western blot analysis..................................4
• 5. Scanning electron microscopy (SEM)......................................................5
• 6. Immunofluorescence staining.......................................................................5
• 7. Immunization and measurement of CSFV E2 antibody titers..........6
• Ⅲ. RESULTS AND DISCUSSION..........................................................7
• 1. Construction of recombinant baculovirus producing recombinant polyhedra with gE2ΔC...................................................................................7
• 2. Production of recombinant polyhedra with gE2ΔC in baculovirus-infected silkworm larvae.....................................................14
• 3. Immune response of recombinant polyhedra carrying gE2ΔC in a mouse model...................................................................................................23
• Ⅳ. CONCLUSION.....................................................................................26
• REFERENCES.........................................................................................28
• ABSTRACT............................................................................................33