Cloning and expression of brain-specific anion transporter BSAT1 and isolation of monoclonal antibodies to its extracellular fragment.
Brain-specific anion transporter BSAT1 extracellular fragment (451-557) cDNA was cloned in a vector for prokaryotic expression, a producer E. coli strain was obtained, and recombinant extracellular fragment BSAT1(451-557)was purified and used for immunization of BALB/c mice. Splenic cells from mice with verified immune response were used for hybridoma generation. Several hybridoma clones producing monoclonal antibodies to BSAT1 extracellular fragment were selected. Antibody specificity was confirmed by ELISA, immunoblotting with recombinant BSAT1(451-557), and immunofluorescent BSAT1 assay on rat brain sections and cultured HEK293 cells. It was demonstrated that the obtained antibodies specifically bind native rat and human BSAT1 and can be used in both fundamental studies of structures forming the blood-brain barrier and development of targeted transport of diagnostic preparations and drugs across the blood-brain barrier.
Department of Fundamental and Applied Neurobiology, V. P. Serbskii State Research Center of Social and Forensic Psychiatry, Moscow, Russia. serpoff@mail.ru
V P
VP
Baklaushev
K Sh
KSh
Kardashova
O I
OI
Gurina
A V
AV
Leopol'd
A V
AV
Semenova
I A
IA
Ryabukhin
V P
VP
Chekhonin
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007114:Immunization; D007118:Immunoassay; D051379:Mice; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D027701:Organic Cation Transport Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D027361:Organic Anion Transporters; D027701:Organic Cation Transport Proteins; D011994:Recombinant Proteins; C451262:Slco1c1 protein, rat