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Updated: Aug 24, 2026
Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells
Published on: June 5, 2015
[Cloning, prokaryotic expression of CML66 and preparation of its polyclonal antibody]
Qiang You1, Lin-ling Cheng, Ying Wang
1Shanghai Institute of Immunology, Shanghai Second Medical University, Shanghai 200025, China. youqiang@etang.com
Aim:
To clone and express CML66 cDNA and to prepare rabbit anti-CML66 antibody.
Methods:
cDNA isolated from the testis using RT-PCR was cloned into pGEMT. After sequencing, the cDNA was inserted into prokaryotic expression vector pET32b(+). The recombinant vector was transformed into BL-21(DE3) through electroporation. 6xHis-tagged CML66 expression was then induced by IPTG. The protein was purified through Ni(2+) affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified protein was injected into rabbits to prepare polyclonal antibody.
Results:
The cloned cDNA sequence was identical with that previously reported. The target protein was successfully purified. And rabbit's anti-serum with high titer was obtained.
Conclusion:
We have cloned CML66 successfully, expressed and purified the protein in E.coli.Furthermore,rabbit polyclonal antibody has been obtained.
