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Published on: August 9, 2019
[Selection and identification of completely humanized anti-IL-4 single-chain antibodies]
Xinmei Cao1, Siji Nian2, Yingchun Ye2
1Department of Immunology, Preclinic School, Sichuan Medical University, Luzhou 646000, China.
Objective:
To express human interleukin 4 (IL-4), select completely humanized anti-IL-4 single-chain antibodies (scFvs) from the completely humanized scFv library and identify their specificity.
Methods:
With IPTG-induced pET102/IL-4/BL21, human IL-4 was synthesized, and then purified and identified. Completely humanized scFvs against IL-4 were expressed from the completely humanized antibody phage library, and positive clones were selected with human IL-4 as antigen. The positive clones were tested by PCR, enzyme cleavage and sequencing. The specificity of the completely humanized anti-IL-4 scFvs was determined by dot blot hybridization.
Results:
The relative molecular mass (Mr) of IL-4 fusion protein was about 27 000. With IL-4 as antigen, we obtained the positive clones expressing anti-IL-4 antibodies. PCR showed amplified target bands of about 1000 bp. Enzyme cleavage analysis revealed that fingerprint maps of the selected clones were diverse except 2 clones. Among 4 clones expressing scFvs that had different fingerprint maps and strong binding to IL-4, sequencing showed the sequence of 3 was correct. Then, dot blot hybridization demonstrated that the scFvs expressed by the 3 clones were specific.
Conclusion:
Completely humanized scFvs against IL-4 has been obtained successfully.
