[Gene's construction, expression and activity analysis of humanized single-domain antibody against human lung cancer].
OBJECTIVE
To construct the gene of humanized single-domain antibody hu3D3V(H) against human lung cancer, to express it in E.coli and analyze its activity.


METHODS
The mAb3D3V(H) was humanized by using CDRs grafting technique. The hu3D3V(H) gene was assembled by overlapping PCR. The expression vector pET22(b+)/hu3D3V(H) was constructed and transformed into E.coli BL21(DE3) and the hu3D3V(H) protein was expressed under IPTG induction. After purification by Ni(2+)-affinity chromatographic column, the activity of hu3D3V(H) protein was analyzed by indirect ELISA and competitive inhibition ELISA.


RESULTS
The target gene with correct sequence was obtained by overlapping PCR. The hu3D3V(H) protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial proteins. After purification, the purity of hu3D3V(H) was more than 95%. The reactivity of purified protein was the same as parent antibody, and could inhibit the binding of mAb3D3 to L342 cells.


CONCLUSIONS
The hu3D3V(H) still retains the reactivity and specificity of mAb3D3, which lays the foundation for its further clinical application.
Wang
Jie-ping
JP
School of Life Science, Xiamen University, Xiamen 361005, China.
Zhuang
Guo-hong
GH
Yang
Gui-wang
GW
Wang
Zhen
Z
Li
Wen-zhu
WZ
Xie
Lian-ying
LY
Yan
Jiang-hua
JH
D000906:Antibodies / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology*; D008175:Lung Neoplasms / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D000906:Antibodies; D000912:Antibodies, Neoplasm; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins