Cloning and Expression of Gonadotropin-releasing Hormone to Develop an Immunological-based Sterilization Vaccine

Abstract

Background: Gonadotropin hormone-releasing hormone (GnRH) is a peptide involved in mammals’ fertility and may also serve as a candidate target for cancer immunotherapy. Immunonsterilization is known as a proper alternative to surgical castration and has been advocated by European countries in recent years. Immunization with GnRH can effectively inhibit the secretion of gonadotropins and cause infertility in both genders of mammals. In this study, a recombinant trimer of GnRH is designed and expressed in a prokaryotic system. Materials and Methods: A construct containing GnRH trimer was designed and prepared using gene synthesis. A cloning site was embedded and connected to the GnRH using a linker to further clone any protein of interest. The construct was subcloned into a pET-32a+ plasmid vector. The recombinant vector was transferred to BL21, an Escherichia coli strain, and the expression was induced using isopropyl β- d-1-thiogalactopyranoside (IPTG). The cell lysate was prepared using lysis buffer and nickel affinity chromatography purification. The GnRH expression was evaluated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after protein purification. Results: The cloning was verified using a polymerase chain reaction (PCR) followed by sequencing the recombinant vector. The result of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis verified the recombinant protein’s expression and the purification process’s function. Conclusion: The GnRH was properly cloned and expressed in BL21. The results also verified the reliability of the purification process. The construct design allows the researchers to express another peptide sequence attached to the GnRH using the embedded linker to improve the stability and antigenicity. A stable recombinant GnRH would be applied in immunocastration and cancer immunotherapies.