Construction of a chimeric shuttle plasmid via a heterodimer system: secretion of an scFv protein from Bacillus brevis cells capable of inhibiting hemagglutination.
Passive immunization is an attractive therapy for preventing oral diseases including dental caries and periodontal disease. For this purpose, we attempted to produce a single chain variable fragment, scFv, which inhibited hemagglutination using the Bacillus brevis protein-producing system. To accomplish this, a novel strategy, a heterodimer system, was used for the construction of a chimeric shuttle plasmid. Initially, a set of new plasmids, kanamycin-resistant donor and erythromycin-resistant general cloning plasmids, were constructed. p15A ori was a common replication origin in these plasmids, while the pUB110 rep and minus origin (MO) were cloned into the donor plasmid. Next, the secretion domain of the B. subtilis alpha-amylase gene and the G2-4 gene, coding for the scFv protein, were cloned into the general cloning plasmid and fused by PCR. Both the donor plasmid and the general cloning plasmid containing the fused gene were digested with NotI and them ligated, a dimeric plasmid being constructed. The key restriction sites, AscI, are arranged such that the pUB110 rep-MO moiety was switched from the donor to the general cloning plasmid following AscI digestion. The chimeric shuttle plasmid was readily constructed by simple re-circularization and a B. brevis transformant producing the scFv protein in the culture fluid was isolated.
Shiroza
T
T
Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan. tshiroza@mascat.nihon-u.ac.jp
Shibata
Y
Y
Hayakawa
M
M
Shinozaki
N
N
Fukushima
K
K
Udaka
S
S
Abiko
Y
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics*; D001483:Base Sequence; D002678:Chimera / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D003731:Dental Caries / Q000517:prevention & control; D005798:Genes, Bacterial; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D007116:Immunization, Passive; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010510:Periodontal Diseases / Q000517:prevention & control; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics
D017931:DNA Primers; D004274:DNA, Recombinant; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases