[Construction of scFv antibody mini-library to 3 cellular surface molecules].
To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin (N-cad), CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p III protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The cDNA of VH and VL genes were amplified by RT-PCR, and a scFv-phage display antibody library was constructed with the amplified V-genes. The content, multiplicity and expressing potential of the library were examined. As a result, we had produced a scFv library containing 1.4 x 10(6) individual clones which showed different patterns after being digested with restriction endoneuclease Mua I. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133. The library will be useful for isolating corresponding specific scFv antibodies.
Huang
Yuelong
Y
Reproduction and Stem Cell Engineering Institute of Central South University, Changsha 410078, China.
Li
Xia
X
Xu
Jing
J
Liu
Junxia
J
Song
Zengxuan
Z
Lu
Guangxiu
G
D000071916:AC133 Antigen; D000818:Animals; D000906:Antibodies / Q000235:genetics*; D015703:Antigens, CD / Q000276:immunology; D018952:Antigens, CD34 / Q000276:immunology; D001483:Base Sequence; D015820:Cadherins / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000276:immunology
D000071916:AC133 Antigen; D000906:Antibodies; D015703:Antigens, CD; D018952:Antigens, CD34; D015820:Cadherins; D006023:Glycoproteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D010455:Peptides; C000606024:Prom1 protein, mouse; C055966:immunoglobulin Fv