Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli.
The Pseudomonas aeruginosa lipoprotein gene (oprI) was modified by cloning an in-frame polylinker in both orientations at the end of oprI. The resulting plasmids pVUB1 and pVUB2 allow high lipoprotein production in E. coli after IPTG induction. The modified lipoproteins are present in the outer membrane and surface-exposed. Outer membrane-bound fusion proteins of different sizes were produced and used to generate antibodies without use of adjuvant. An 87 bp DNA fragment from the vp72 capsid protein gene of African Swine Fever virus (ASFV) and the entire Leishmania major glycoprotein gp63 gene were expressed in this system. Finally, a fusion lipoprotein containing a 16 amino acid epitope from the pre-S2b region of Hepatitis B virus (HBV) was presented by an antigen-presenting cell line to a T-cell hybridoma while the corresponding cross-linked S2b peptide was not. The results suggest that OprI-based fusion proteins can be used to generate both humoral and cellular immune responses.
Cornelis
P
P
Laboratorium Algemene Biologie, Vrije Unviersiteit Brussel Vlaams Instituut Biotechnologie, Belgium. pcornel@vub.ac.be
Sierra
J C
JC
Lim
A
A
Malur
A
A
Tungpradabkul
S
S
Tazka
H
H
Leitão
A
A
Martins
C V
CV
di Perna
C
C
Brys
L
L
De Baetseller
P
P
Hamers
R
R
D000358:African Swine Fever Virus / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014614:Vaccines, Synthetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C060507:outer membrane lipoprotein I, Bacteria