title: The expression system of biologically active canine interleukin-8 in Leishmania promastigotes.
abstract: It has been reported that Leishmania promastigotes have ability to express foreign genes on drug selectable plasmids. To investigate further abilities of the recently described expression vector, P6.5, in the transfection of Leishmania organisms (Chen D-Q, Kolli BK, Yadava N et al. Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 levels in promastigotes and their infection of macrophages in vitro. Infect Immun 2000;68:80--86), the constructed expression vector, which contains canine interleukin-8 (cIL-8) coding cDNA, was introduced by electroporation to promastigotes of four species of the genus Leishmania: Leishmania amazonensis, L. equatorensis, L. donovani and L. infantum. Extrachromosomal DNAs and total RNAs from the transfected promastigotes were subjected to polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively, using cIL-8 gene specific primers, and a predicted product of 330 bp was detected. Western blot analysis using a mouse monoclonal antibody raised against cIL-8 demonstrated the successful expression of cIL-8 in the transfectants and culture supernatants. Culture supernatants of the transfected L. amazonensis and L. equatorensis promastigotes showed a high chemotactic activity to both dog and mouse polymorphonuclear leukocytes. These results indicate that Leishmania promastigotes transfected with the expression vector P6.5 containing cIL-8 cDNA are capable of producing biologically active cIL-8. The Leishmania expression system using the P6.5 vector might be a useful alternative for the production of biologically active recombinant cytokines.
authors.affiliation: Department of Molecular Immunology, School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Toshimitsu
authors.initials: T
authors.lastname: Hatabu
authors.forename: Yasunobu
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Kawazu
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Tsugihiko
authors.initials: T
authors.lastname: Kamio
authors.forename: Hong Gang
authors.initials: HG
authors.lastname: Lu
authors.forename: Kwang-Poo
authors.initials: KP
authors.lastname: Chang
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hashiguchi
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kano
authors.forename: Takashi
authors.initials: T
authors.lastname: Onodera
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Matsumoto
mesh_terms: D000818:Animals; D002634:Chemotaxis, Leukocyte; D018076:DNA, Complementary; D004285:Dogs; D005260:Female; D005822:Genetic Vectors; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D007891:Leishmania / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D007963:Leukocytes, Mononuclear; D051379:Mice; D008807:Mice, Inbred BALB C; D011995:Recombination, Genetic; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D016209:Interleukin-8