The Expression, Purification Of MPem Protein In E.coli And The Preparation Of Its Antiserum

The Pem cDNA encodes a polypeptide of 210 amino acids. Residues 116 to 175 resemble a homeodomain. Pem gene is expressed in a time and stage specific manner. Pem transcripts are detectable as early as day 6 in mouse embryonic development, and are abundant in day 7 and 8, but decrease precipitously thereafter. Although Pem transcripts are not detectable in most of adult tissues, they are present in reproductive system. Furthermore, Pem has also shown to be specifically expressed in the proximal cauda and distal corpus regions of the epididymis, the regions where spermatozoa gain forward motility and fertilization competence. These data implicate a important role for Pem during embryogenesis and in sperm development.Using GAL4 yeast two-hybrid system to screen a 7-day mouse embryo library, we had demonstrated that specific gene products interact with mPem protein, including Cdc37 and Mdfic. In a effort to look into the functions of mPem protein and its interactions with other proteins, we prepared the polyclonal antiserum of a recombinant antigen^mPem-6His. Polyclonal antiserum is a useful tool in characterization of mPem product. It can be used directly to detect mPem and its recombinant proteins, to do in vitro studies of mPem function.Object: overexpression of mPem-6His protein in E. coli; getting specific polyclonal antiserum of mPem-6His protein.Methods: The oligonucleotide primers were designed and used to amplify the mPem cDNA fragment. The amplified fragment was then cloned into E.coli expression vector pET22b(+) to construct recombinant plasmid. The recombinant plasmid was transformed into E. coli expression strain Rosetta?2(DE3). IPTG induces the expression protein mPem-6His protein, and the induction conditions were optimized. The induced product was purified by Ni²⁺ affinity chromatography, and the purified product was ultrafiltered to desalt and then lyophilized for future use. The lyophilized product was used as antigen, injected intradermally to immunize male New Zealand white rabbit to get polyclonal antiserum. The titer and the specificity of the rabbit antiserum were detected by ELISA and Western blotting, respectively.