Construction of a chimeric shuttle plasmid via a heterodimer system: Secretion of an scFv protein from Bacillus brevis cells capable of inhibiting hemagglutination

Citation
T. Shiroza et al., Construction of a chimeric shuttle plasmid via a heterodimer system: Secretion of an scFv protein from Bacillus brevis cells capable of inhibiting hemagglutination, BIOS BIOT B, 65(2), 2001, pp. 389-395
Citations number
17
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Agricultural Chemistry","Biochemistry & Biophysics
Journal title
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
ISSN journal
0916-8451 → ACNP
Volume
65
Issue
2
Year of publication
2001
Pages
389 - 395
Database
ISI
SICI code
0916-8451(200102)65:2<389:COACSP>2.0.ZU;2-3
Abstract
Passive immunization is an attractive therapy for preventing oral diseases including dental caries and periodontal disease. For this purpose, we attem pted to produce a single chain variable fragment, scFv, which inhibited hem agglutination using the Bacillus brevis protein-producing system. To accomp lish this, a novel strategy, a heterodimer system, was used for the constru ction of a chimeric shuttle plasmid. Initially, a set of new plasmids, kana mycin-resistant donor and erythromycin-resistant general cloning plasmids, were constructed. p15A ori was a common replication origin in these plasmid s, while the pUB110 rep and minus origin (MO) were cloned into the donor pl asmid. Next, the secretion domain of the B. subtilis alpha -amylase gene an d 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 cl oning plasmid containing the fused gene were digested with Not1 and them li gated, a dimeric plasmid being constructed. The hey restriction sites, Asci , are arranged such that the pUB110 rep-MO moiety was switched from the don or to the general cloning plasmid following Asci digestion. The chimeric sh uttle plasmid was readily constructed by simple re-circularization and a B. brevis transformant producing the scFv protein in the culture fluid was is olated.