Expression of apxIA of Actinobacillus pleuropneumoniae in Saccharomyces cerevisiae.
- Author:
Sung Jae SHIN
1
;
Jong Lye BAE
;
Young Wook CHO
;
Moon Sik YANG
;
Dae Hyuk KIM
;
Yong Suk JANG
;
Han Sang YOO
Author Information
1. Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul, 151-742, Korea. yoohs@plaza.snu.ac.kr
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
Actinobacillus pleuropneumoniae;
apxIA;
expression;
Saccharomyces cerevisiae
- MeSH:
Actinobacillus pleuropneumoniae/*genetics/isolation & purification/metabolism;
Animals;
Bacterial Proteins/*biosynthesis/genetics;
Blotting, Western/veterinary;
Cloning, Molecular;
DNA, Bacterial/chemistry/genetics;
Electrophoresis, Polyacrylamide Gel/veterinary;
Hemolysin Proteins;
Pleuropneumonia, Contagious/microbiology;
Polymerase Chain Reaction/veterinary;
Saccharomyces cerevisiae/genetics/*metabolism;
Swine;
Swine Diseases/microbiology
- From:Journal of Veterinary Science
2003;4(3):225-228
- CountryRepublic of Korea
- Language:English
-
Abstract:
Actinobacillus pleuropneumoniae is an important primary pathogen in pigs, in which it causes a highly contagious pleuropneumoniae. In our previous study, apxIA gene amplified from A. pleuropneumoniae Korean isolate by PCR with primer designed based on the N- and C-terminal of the toxin was cloned in TA cloning vector and sequenced. The nucleotide sequences of apxIA gene was reported to GeneBank with the accession numbers of AF363361. Identity of the Apx IA from the cloned gene in E. coli was proved by SDS-PAGE and Western blot. Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics and vaccine productions. Therefore, to use the yeast as a delivery system in new oral subunit vaccine, apxIA gene was subcloned into Saccharomyces cerevisiae, and ientified the expression of Apx IA protein. First, apxIA gene was amplified by PCR with the primers containing BamHI and SalI site at each end. Second, the DNA digested with BamHI and SalI was ligated into YEpGPD-TER vector, and transformed into S. cerevisiae 2805. Third, after identification of the correctly oriented clone, the 120-kDa of Apx IA protein expressed in S. cerevisiae 2805 was identified by SDS-PAGE and Western blot.
