title: Identification and characterization of two CD4 alleles in Microminipigs.
abstract: BACKGROUND
  We previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies.
  
  
  RESULTS
  We identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation.
  
  
  CONCLUSIONS
  We identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis.
authors.affiliation: United Graduate School of Veterinary Sciences, Gifu University, Gifu, 501-1193, Japan.|Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Matsubara
authors.affiliation: United Graduate School of Veterinary Sciences, Gifu University, Gifu, 501-1193, Japan. nishii@gifu-u.ac.jp.|Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. nishii@gifu-u.ac.jp.
authors.forename: Naohito
authors.initials: N
authors.lastname: Nishii
authors.affiliation: Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takashima
authors.affiliation: United Graduate School of Veterinary Sciences, Gifu University, Gifu, 501-1193, Japan.|Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Takasu
authors.affiliation: Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Noriaki
authors.initials: N
authors.lastname: Imaeda
authors.affiliation: Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Kayo
authors.initials: K
authors.lastname: Aiki-Oshimo
authors.affiliation: United Graduate School of Veterinary Sciences, Gifu University, Gifu, 501-1193, Japan.|Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Yamazoe
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, 351-0198, Japan.
authors.forename: Michinori
authors.initials: M
authors.lastname: Kakisaka
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, 351-0198, Japan.
authors.forename: Shin-Nosuke
authors.initials: SN
authors.lastname: Takeshima
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, 351-0198, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Aida
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, 259-1193, Japan.
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Kametani
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, 259-1193, Japan.|School of Psychiatry and Clinical Neurosciences, The University of Western Australia, Crawley, WA, 6009, Australia.
authors.forename: Jerzy K
authors.initials: JK
authors.lastname: Kulski
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, 259-1193, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Ando
authors.affiliation: United Graduate School of Veterinary Sciences, Gifu University, Gifu, 501-1193, Japan.|Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kitagawa
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D014644:Genetic Variation; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000235:genetics / Q000378:metabolism*; D013556:Swine, Miniature / Q000235:genetics / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; D012333:RNA, Messenger
keywords: Amino-acid substitution; CD4 polymorphism; Microminipigs; PCR-RFLP