Expression And Purification Of PET32a-XBP1u Protein And Preparation Of Polyclonal Antibody Against Protein XBP1u

ObjectivesIn eukaryocyte, X-box binding protein 1 (XBP1) has many biological function as a transcriptional activator, it has wide biology effect, participate in many physiological and pathological procession. There has been many focus on the influence of XBP1 on diseases. XBP1 has two forms and XBP1u is its unspliced style, which has transcriptional activity, too. In this study, we make use of molecular biologic technology just as the polymerase chain reaction (PCR),gene cloning and so on, to construct the prokaryotic expression plasmid pET32a-XBP1u. The recombinant plasmid was induced expression in E.coli BL2l, then we got lots of XBP1u protein with cytoryctes, expression. Furthermore, the purified XBP1u protein was generated by using Ni-NTA affinity chromatography, and we use Western blot to analysis our interest protein. The successful production of purified and exact XBP1u protein was used to immunize the rabbits, then the polyclonal antibody was gained. This will be a basis of further studying of structure and biological function of XBP1u . Methods(1) Abstraction of total RNA from hepatoma carcinoma cell (HepG2) and two steps , RT-PCR was used to amplify the target gene XBP1u.(2) XBP1u was inserted into the pGEM-T vector by using molecular cloning technology to construct plasmid pGEM-T -XBP1u.(3) Construct the prokaryotic expression plasmid pET32a-XBP1u by using molecular cloning technology.(4) The prokaryotic expression plasmid pET32a-XBP1u was induced by IPTG in E.coli BL2l, we got the interest protein. The interest protein was analyzed by SDS-PAGE and purified by Ni2+-NAT chromatography. Its protein concentration was detected by BCA methods.(5) We use Western blot to analysis whether our interest protein was exact.(6) The target protein was used to immunize the rabbits.(7) The titer of the antiserum was detected by ELISA.(8) The specificity of the rabbits, antiserum was detected with Western blot.Results(1) The target gene XBP1u was amplified, and the interest gene was sequenced as 785bp, the interest protein encoded polypeptides of 261 amino acid residues.(2) The prokaryotic expression plasmid XBP1u has been constructed successfully, and transformed it into E.coli BL2l.(3) The expression of pET32a-XBP1u was induced by IPTG, SDS-PAGE showed that the interest fusion protein of XBP1u mainly expressed as inclusion bodies. After purified, the purity of target protein was over 90% and its concentration was 1.42mg/ml.(4) Western blot to analysis the antigenicity of interest protein, and the interest protein can do immune reaction with the His tag antibody. Due to pET32a of pET32a-XBP1u has His tag on it, we can use anti-His antibody and anti-XBP1antibody to detect our interest protein, then we can conclude that this interest protein was XBP1u.(5) The titer of the antiserum was 1:64000.(6) The specified band was found in Western blot.ConclusionsThe prokaryotic expression plasmid XBP1u has been constructed successfully, and transformed it into E.coli BL2l, then we got lots of XBP1u protein, it has higher purity. We gained the polyclonal antibody of XBP1u. This will be a basis of further studying of structure and biological function of XBP1u.