Construction And Expression Of Recombinant Extracellular Domain Of Human ICOS In Escherichia Coli

The experiment is to construct and express prokaryotic expression vector of recombinant extracellulardomain gene of human inducible co-stimulator (ICOS). And then use the purified protein as antigen to immunize mice. Primers were designed according to the reference extracellular domain of human ICOS gene from GenBank. The extracellular domain gene was amplified from total mRNA of human T-cell lymphoma cell line Jurkat by using RT-PCR. The extracellular homodimer gene of human ICOS was amplified by using overlap-extension PCR. The PCR product was cloned into the T vector and sequenced. The homodimer gene was inserted into pET-28a plasmid, and identified by PCR, digesting and sequencing analysis. Target protein was distinctly expressed after transformed into E. coli BL21 (DE3) and induced with IPTG. The target protein was purified by denaturation and renaturation from inclusion body, and was detected by Western blot. Then the purified protein was used as antigen to immunize mice.The sequencing result showed that the target gene of extracellular homodimer of human ICOS was 757 bp, and the recombinant plasmid was constructed correctly. By the challenge of SDS-PAGE and Western blot analysis showed that the target protein was over-expressed in E. coli BL21 (DE3), and its molecule weight was about 31 KD. It is as the same with the expected results. And get a better mice immunized with antibody titer. Prokaryotic expression vector of recombinant extracellular domain of human ICOS has been achieved and was over-expressed in E. coli BL21 (DE3) successfully, which provides a basis for the next protein purification and the specific anti-ICOS antibody preparation.