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.
We developed 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, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. 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 as an antigen delivery system. 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.
Microbe-Based Fusion Technology Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Jeonbuk 580-185, South Korea.
J O
JO
Baek
J W
JW
Seo
O
O
Kwon
S M
SM
Park
C H
CH
Kim
I H
IH
Kim
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006801:Humans; D007114:Immunization; D007371:Interferon-gamma / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D046529:Microscopy, Electron, Transmission; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism; D030361:Papillomavirus Infections / Q000517:prevention & control; D053918:Papillomavirus Vaccines / Q000008:administration & dosage / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000517:prevention & control*; D014771:Virion / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure
D000914:Antibodies, Viral; D036022:Capsid Proteins; C114580:HPV L1 protein, Human papillomavirus; D009856:Oncogene Proteins, Viral; D053918:Papillomavirus Vaccines; D011994:Recombinant Proteins; D007371:Interferon-gamma