Cloning, expression, purification and identification of CCR5 extracellular domain recombinant protein

    WU Kongª²Tian, ZHANG Yingª²Qi£¬ YAN Zhen

    Biotechnology Center, School of Pharmacy, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct a prokaryotic expression vector of CCR5 extracellular domain recombinant protein and to purify and identify it. METHODS: To obtain amino acid sequence of CCR5 extracellular domains, the four peptides of CCR5(Nª²term, ECL1, ECL2 and ECL3)were combined using the soft linker and named rCCR5. RCCR5 gene was synthesized according to codon E.coli preferred. The gene was cloned into the vector pETª²22b(+) and the aim protein was expressed in E.coli. The expression product was purified and then identified with Westernª²blot. RESULTS: We developed a purification process of rCCR5 from inclusion bodies. The procedure included washing and solubilization of inclusion body, purification by SephacrylSª²300 and refolding by SephacrylSª²100. The result of Westernª² blot showed the specific binding of CCR5 mAb with aim protein. CONCLUSION: Purified recombinant protein rCCR5 is obtained successfully, which can be used as a candidate antigen for CCR5 autovaccine to challenge HIVª²1 infection. ......