[Construction and expression of single chain Fv antibody against human bladder carcinoma].
OBJECTIVE
To construct and express single chain Fv antibody against human bladder carcinoma, which was expected to have advantages in targeted diagnosis and therapy with the characteristics of lower immunogenicity.


METHODS
Hybridoma BDI-1 cell, which secreted a monoclonal antibody against human bladder carcinoma, was used to isolate total RNA. By reverse transcription, the cDNA was synthesized and used as templates for amplifying the immunoglobulin heavy-and light-chain variable region genes by polymerase chain reaction (PCR). The amplified DNA was ligated into a sequencing vector pUC19 and sequenced with Sanger's method. The VH and VL genes were inserted into expression vector pFUW80. By inducing, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected by ELISA. The 5 x his-tagged ScFv antibodies were purified on IDA-Ni2+ resin by immobilized metal chelate affinity chromatography (IMAC). The purified ScFv antibodies were analyzed by SDS-PAGE.


RESULTS
A full-length of VH and VL genes was 366 and 324 base pairs respectively. Comparing with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from re-arrangement of VH, Dsp2.2 and JH4; the VL gene was VK subgroup IV and from Vk and Jk4. The ScFv antibodies could inhibit 84% of the antigen binding activity of original McAb BDI-1. The purified ScFv antibodies gave a single major band (Mr-29 000) on SDS-PAGE.


CONCLUSIONS
The single chain Fv antibody against human bladder carcinoma was successfully constructed and expressed.
Zhang
M
M
Institute of Urology, Peking University, Beijing 100034, China.
Yu
L
L
Huang
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D051379:Mice; D010957:Plasmids; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D001749:Urinary Bladder Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins