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[Gene's construction, expression and activity analysis of humanized single-domain antibody against human lung cancer]
Jie-ping Wang1, Guo-hong Zhuang, Gui-wang Yang
1School of Life Science, Xiamen University, Xiamen 361005, China.
Aim:
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.
Conclusion:
The hu3D3V(H) still retains the reactivity and specificity of mAb3D3, which lays the foundation for its further clinical application.
