Expression of Recombinant bovine L-selectin in Escherichia coli and insect cells.
Bovine L-selectin was expressed in bacteria using pGEX vector and in insect cells infected with recombinant baculovirus in order to obtain recombinant protein for preparation of specific antiserum and its functional studies. In bacterial expression, L-selectin fusion protein with glutathione S-transferase was detected in the insoluble fraction with the expected molecular weight of 60 kDa by SDS-PAGE and reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. In insect cells infected with recombinant baculovirus, a band corresponding to L-selectin was not observed in SDS-PAGE with protein staining, but they apparently reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. Furthermore, the indirect immunofluorescence test revealed that bovine L-selectin was efficiently expressed on the surface of Sf9 cells infected with recombinant baculovirus, and flow cytometric analysis showed that the percentage of CD62L positive cells in bovine PBMC was about 66% and that most Sf9 cells infected with recombinant baculovirus had specific immunofluorescence.
Kashima
T
T
Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Japan.
Hasegawa
A
A
Iwata
H
H
Inoue
T
T
D000818:Animals; D002417:Cattle / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D019084:Fluorescent Antibody Technique, Indirect; D007106:Immune Sera; D019041:L-Selectin / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera
D007106:Immune Sera; D011994:Recombinant Proteins; D019041:L-Selectin