[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A].
The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.
Zhao
B H
BH
College of Life Science, Hebei Normal University, Shijiazhuang 050016, China.
Xu
C B
CB
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000276:immunology*; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000276:immunology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017422:Sequence Analysis, DNA; D010738:Type C Phospholipases / Q000276:immunology*
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens