[Construction and expression of anti-GD2/anti-CD16 single-chain bispecific antibody].
This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody(sc-BsAb), and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene (NM3E2) were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b(+) plasmid from the pMD18-T easy vector by digestion with NcoI, Hind III restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 (DE3). The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni2+ -NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusion protein is SerGly4Ser. and the molecular is 53 KD.
Ni
Jianfeng
J
Group of Microbiological Engineering,Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China.
Ji
Jianfei
J
Bai
Xiangyang
X
Lu
Anguos
A
Huang
Ribo
R
Wei
Yutuo
Y
Wu
Wenfang
W
D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000235:genetics; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005732:Gangliosides / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D008545:Melanoma / Q000473:pathology; D008969:Molecular Sequence Data; D017452:Receptors, IgG / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D018033:Antibodies, Bispecific; D000912:Antibodies, Neoplasm; D005732:Gangliosides; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; C019403:ganglioside, GD2