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| Prokaryotic Expression of Human CD24 and Its Polyclonal Antibody Preparation |
| LIN Jie1, MA Lan1,2, ZHANG Shi-yi1, GUAN Chang-dong1, LI Xuan1, LV Qi1 |
1. The Monoclonal Antibody Engineering and Technology Centre, School of Life Science, Yunnan University, Kunming 650091, China;
2. Life Science Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China |
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Abstract Human CD24 is a candidate biomarker specifically expresses in many types of malignant tumor cells. In order to obtain its polyclonal antibody for following-up studies, the encoding sequence of mature human CD24 polypeptides without glycosylation (hCD24N) was amplified and cloned into pGEX-KGV, which is a modified prokaryotic expression vector containing a N-terminus GST fusion tag and another internal Strap tag II. Prokaryotic expression plasmid pGEX-KGV-hCD24N was constructed and expressed in the strain of E. coli BL21 (DE3) using lactose induction. Fusion protein, named GST-StraptagII-hCD24N, mainly expressed in its soluble forms, and existed in the supernatant after cell lysis. Using GST affinity chromatographic separation, this target protein was purified and used to immunize rabbit to produce rabbit anti-hCD24N polyclonal antibody. An indirect ELISA assay was further used to measure the anti-hCD24N titer of prepared antibody. Moreover, the specificity against native glycosylated CD24 molecular which is expressed in the positive human cancer cells was also confirmed by Western blot and immunofluorescence, and compared with commercial antibody. The results showed that the prepared polyclonal antibody had a high titer to recombinant hCD24N, and could react with native human CD24 and its positive human malignant cancer cells specifically, which provides a favorable tool for further studies on application of CD24 as a candidate antigen for cancer prevention and treatment. Moreover, the GST-StraptagII-hCD24N fusion protein could also be used as a target for screening novel specific ligand against CD24 and associated positive cancer cells. In conclusion, the prepared anti-CD24N polyclonal antibody provides the basis for tumor biology research and related diagnostic reagents development which are targeting CD24 molecular.
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Received: 15 August 2011
Published: 25 December 2011
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