Of Human prothymosin α and interleukin-2 fusion protein gene cloning and prokaryotic expression of.docVIP

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2017-05-05 发布于浙江
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Of Human prothymosin α and interleukin-2 fusion protein gene cloning and prokaryotic expression of.doc

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Of Human prothymosin α and interleukin-2 fusion protein gene cloning and prokaryotic expression of

Of Human prothymosin α and interleukin-2 fusion protein gene cloning and prokaryotic expression of [Abstract] Objective To clone human prothymosin α / IL-2 fusion gene construct its prokaryotic expression vector and expressed in E. coli system effectively. Methods RT  PCR method from human leukemia cells and human T lymphocytes, respectively, amplified by prothymosin α / IL-2 gene, using recombinant technology to fusion gene fragment in pET42a prokaryotic expression recombinant vector construct into a pET42a  PI. After enzyme digestion, DNA sequencing, the recombinant plasmid was transformed into E. coli Rosseta, IPTG-induced protein expression. Expressed protein purified by ion-exchange and molecular sieve after Western blot analysis of human peripheral blood mononuclear cells by rosette experiments fusion protein had a preliminary determination of the activity. The results of gel electrophoresis-PCR product of a length of about 750 bp, sequencing results showed that the sequence of amplified fragments in line with expectations. IPTG-induced recombinant bacteria to express the relative molecular mass of 28 kDa protein, the purified fusion protein can enhance human peripheral blood mononuclear cells in rosette formation rate, indicating that a certain activity. Conclusion We successfully cloned and building of human prothymosin α / IL-2 fusion gene in the prokaryotic expression vector and prokaryotic expression system to be effectively expressed. [Keywords:] prothymosin α ; interleukin-2; fusion protein; expression Abstract: Objective To clone human prothymosin α and interleukin  2 fusion gene into the prokaryotic expression vector and to express the fusion protein effectively in E.coli system.Methods The human prothymosin α and interleukin  2 gene were amplified by RT  PCR from human leukemia cells and T lymphocytes respectively. The two genes were fused with a linker and the fusion gene fragment was used to construct a prokaryotic expression vec