The SUMO Fusion System Efficiently Expresses and Prepare Polyclonal Antibody the Soluble Helicobacter Pylori Ferritin Protein
Helicobacter pylori is a protein in which iron is stored in the body and maintains iron balance in the body.It is found in different organisms,but the homology of the ferritin amino acid sequence is low,and Hp is established by using ferritin,which provide a theoretical basis for molecular biology and immunological detection methods.In order to obtain soluble Hp ferritin with SUMO fusion system,primers were designed based on Hp ferritin gene sequence in GenBank.The Hp ferritin gene fragment was amplified by PCR to construct a recombinant clone strain.Positive clones were screened by bacterial liquid PCR,double enzyme digestion,and sequence determination.After double enzyme digestion,the target gene was recovered and a re-combinant expression plasmid pET-28a-SUMO Ferritin was constructed,which was then transformed into DH5α.The positive recombinant expression strain pET-28a-SUMO-Fertitin/BL21 was obtained through bacterial liquid PCR,double enzyme diges-tion,and sequence determination.The recombinant protein was identified by IPTG induction,SDS-PAGE,and Western blotting,and purified using a nickel ion chelating column(Ni NTA).Then,the fusion protein was cleaved using SUMO protease I to obtain high-purity target protein.Finally,the obtained high-purity recombinant protein was used to immunize mice and prepare multi antibodies.Finally,polyclonal antibodies were prepared by immunizing mice with the obtained high-purity recombinant protein.The results showed that an Hp ferritin gene of 501 bp in length was amplified.The recombinant prokaryotic expression plasmid was induced by IPTG.SDS-PAGE showed that there was a band at 34 kDa which was consistent with the theoretical value.Western blotting was used to identify the SUMO fusion recombinant protein,which can specifically bind to His antibod-ies.37 ℃ and 0.1 mM IPTG was induced overnight,and the fusion SUMO recombinant protein was soluble.After removing the SUMO tag with SUMO protease,the recombinant protein was purified and dialyzed to obtain high-purity recombinant protein.The antibody level obtained was 1:25 600.The results show that the soluble Hp ferritin is efficiently expressed by the SUMO fusion system and the polyclonal antibodies were prepared,which lays a foundation for further obtaining monoclonal antibodies and establishment of Hp immunological detection methods.
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