Objective To construct a recombinant bacterium that expresses the specific gene Mmc-3740 of Mycoplasma mycoides subsp. capri for further study and application of the protein.
Method Mmc-47 was amplified by PCR, then cloned and sequenced. The cloned plasmids and pET-28a were undergone a double digestion to recover the correct fragments for ligation and construction of plasmid with the recombinant expression. Verified by sequencing, the plasmid was transformed into the expression strain, BL21 (DE3). The expression was induced using IPTG to obtain the products to be identified by SDS-PAGE and purified through the nickel affinity chromatography to produce hyperimmune serum against mouse. The serum was then identified by the western-blot for confirmation.
Results The cloning and expression vectors were successfully established. The molecular weight of the recombinant protein was approximately 21 ku. The expressed product was purified showing an apparent immunogenicity.
Conclusion The Mmc-3740 recombinant protein was successfully expressed in BL21 (DE3) to provide the basis for further study on the functions and characteristics of the gene.
DownLoad: