Production, Purification, and Characterization of Human Papillomavirus L1 Major Capsid Protein and Virus-like Particles in Sf-9 Cell and Bacillus subtilis for a Cervical Cancer Vaccine
- 주제(키워드) Cervical Cancer , Virus-like Particle , Insect Cell
- 발행기관 고려대학교 대학원
- 지도교수 김익환
- 지도교수 김철호
- 발행년도 2012
- 학위수여년월 2012. 8
- 학위구분 박사
- 학과 일반대학원 생명공학과
- 세부전공 생물법제학
- 원문페이지 124 p
- 실제URI http://www.dcollection.net/handler/korea/000000034683
- 본문언어 영어
- 제출원본 000045717830
초록/요약
This paper is consisting of total three chapters about vaccine candidate study against a cervical cancer. For the first chapter, major capsid protein coding L1 genes of HPV high-risk types 16, 18, 33, and 58 previously isolated from Korean patients were expressed in Sf-9 insect cells using a baculovirus system. Expression of HPV L1 genes was examined by SDS-PAGE, Western blotting, and ELISA using mouse anti-HPV16 L1 monoclonal antibody. Transmission electron microscopy of immunoactive fractions showed HPV virus-like particles (VLPs) of approximately 50~60 nm diameter, created by self-assembly of HPV L1 proteins. The biological activities of VLPs produced in insect cells were confirmed by hemagglutination, thus showing that the VLPs may be useful in development of a cervical cancer vaccine. On the second chapter, the major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. HPV 33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs. The expression and purification process of HPV33 L1 protein in Sf-9 cells was examined by SDS-PAGE, western-blotting, and ELISA analyses, and the data show that HPV33 L1 VLPs were determined to >98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV33 VLPs were approximately 50~60 nm in diameter and created by self-assembly of HPV33 L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine. On the third chapter, it was developed for a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, it was isolated for self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5-1.0 M NaCl. The HPV33 L1 protein was shown to be approximately 56 kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20-40 nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production from antigen delivery. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.
more목차
ChapterI.Expression of virus-like particles of human papillomavirus high-risk types 16, 18, 33 and 58 derived from Sf-9 insect cells 1
1.Introduction 2
2.Materials and Methods 4
2.1 Cells and viruses 4
2.2 Antibodies 4
2.3 Preparation of recombinant baculoviruses 4
2.4 Expression of HPV L1 and purification of VLPs 7
2.5 Western blot analysis 7
2.6 Indirect ELISA 8
2.7 Hemagglutination (HA) assay 8
2.8 Transmission electron microscopy (TEM) 9
3. Results and Discussion 10
3.1 Isolation of the L1 genes of HPV high-risk types 16, 18, 33, and 58 from Korean patients 10
3.2 Preparation of recombinant baculoviruses expressing HPV L1 genes 12
3.3 Expression of HPV L1 genes in insect cells 14
3.4 Identification of HPV VLPs 16
3.5 Biological activity of HPV VLPs 19
4.Conclusions 21
5.References 22
ChapterII.Purification of human papillomavirus (HPV) type 33 L1 virus-like particles from Sf-9 cells using two-step column chromatography 26
1.Introduction 27
2.Materials and Methods 30
2.1 Expression of HPV33 L1 and purification of VLPs 30
2.2 Western blot analysis 30
2.3 Quantitative analysis by immuno-indirect ELISA 31
2.4 Determination of protein concentration 31
2.5 Strong cation-exchange chromatography 32
2.6 Ceramic hydroxyapatite Type II chromatography 32
2.7 Transmission electron microscopy (TEM) 33
2.8 Ultrafiltration using a membrane filter device 33
3.Results and Discussion 34
3.1 Amplification of recombinant baculovirus expression the HPV33 L1 gene 34
3.2 Expression of HPV33 L1 gene in Sf-9 insect cells 36
3.3 Strong cation-exchange chromatography 38
3.4 Ceramic hydroxyapatite chromatography 42
3.5 Identification of self-assembled HPV33 L1 VLPs 45
3.6 Preparation of a final product for vaccination 47
4.Conclusion 48
5.References 51
ChapterIII.Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer 55
1.Introduction 56
2.Materials and Methods 60
2.1 Strains and culture conditions 60
2.2 Plasmids 60
2.3 Antibodies 60
2.4 Amplification of the HPV33 L1 gene, and cloning thereof 61
2.5 Expression of the HPV33 L1 major capsid protein in B.subtilis 62
2.6 Western blot analysis 62
2.7 Confocal microscopy 63
2.8 Purification of HPV33 L1 protein by 40% (w/v) sucrose cushion centrifugation 64
2.9 Purification of HPV33 L1 protein by strong cation exchange chromatography 64
2.10 Indirect immuno-ELISA 65
2.11 Determination of protein concentration 65
2.12 Transmission electron microscopy (TEM) 66
2.13 Immunization of mice 66
2.14 Antibody production 67
2.15 Enzyme-linked spot (ELISPOT) assay for interferon (IFN)- γ 68
3.Results and Discussion 69
3.1 Amplification of the HPV type 33 L1 gene and construction of an HPV33 L1 expression plasmid 69
3.2 Expression of the HPV33 major capsid protein in the two recombinant B. subtilis strains 72
3.3 Purification of HPV33 L1 protein by sucrose cushion centrifugation and ion exchange chromatography 76
3.4 Transmission electron microscopy (TEM) to visualize VLPs 81
3.5 Humoral and cellular immune responses 83
4.Conclusion 86
5.References 89
Conclusion Remarks 96
Abstract in Korean 98
Appendix 100
Acknowledgement 103
