Cloning,expression and purification of His-tagged mouse gene SRG-S

Objective To construct prokaryotic expression plasmid of His-tagged mouse spermatogenesis related gene SRG-S, further to express and purify the recombinant protein.Methods Total RNA was extracted from mouse testis.The fragment of target gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) and cloned into pGEM-T-easy.After being sequenced,the fragment was subcloned into prokaryotic expression vector pET-30a(+) and transformed into the E.coli BL21(DE3).Then IPTG was used to induce the expression of recombinant protein.The fusion protein was further purified with Ni-NTA affinity chromatography and confirmed with anti-His monoclonal antibody by Western bloting.Results The amplified fragment of the target gene was about 591 bp by RT-PCR and its sequence was the same as that in GenBank.SDS-PAGE demonstrated that the fusion protein could be expressed with molecular weight around 32 kD under the induction of IPTG.The purity was above 90% and Western bloting demonstrated the immune specificity against His of the purified fusion protein.Conclusion The prokaryotic expression vector for His-tagged mouse SRG-S is successfully constructed by gene cloning technique,and the His-tagged fusion protein with high purity is obtained.The present results may be helpful for antibody preparation and further investigation into the function of this gene at protein level.