Expression of apxIA of Actinobacillus pleuropneumoniae in Saccharomyces cerevisiae.
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 GenBank 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 identified 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.
Shin
Sung Jae
SJ
Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul, 151-742, Korea.
Bae
Jong Lye
JL
Cho
Young Wook
YW
Yang
Moon Sik
MS
Kim
Dae Hyuk
DH
Jang
Yong Suk
YS
Yoo
Han Sang
HS
D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D006460:Hemolysin Proteins; D011002:Pleuropneumonia, Contagious / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; C087744:ApxI toxin, Bacteria