title: [Cloning and expression of Oreochromis aurea gonadotropin-releasing hormone cDNA in Escherichia coli and its immunogenicity for mice].
abstract: To study the function of the GnRH protein, the recombinant pMAL-GnRH was constructed and expressed in TB1 E. coli. The cDNA encoding gonadotropin-releasing hormone (GnRH) and GnRH associated peptide (GAP) was amplified from total RNA of O. aurea pituitary glands by reverse transcription polymerase chain reaction (RT-PCR), and then blasted against other GnRH cDNA sequences in the GenBank. The analysis of the sequence data indicated that the coding region of the cDNA fragment, which encoded 89 amino acid residues, was about 400 bp in size. The amplified cDNA fragment was cloned into the prokaryotic expression vector, pMAL-c2x, to produce the expression vector pMAL-GnRH. The recombinant plasmid was transformed into E. coli TB1. GnRH-MBP fusion protein was obtained after the addition of IPTG into the growth media. SDS-PAGE analysis revealed that the GnRH-MBP was expressed after induction with IPTG for 4 h. A protein band of 56 kD appeared on SDS-PAGE gel and was proved by Western blot. The mass production of the recombinant protein was about 41.6% of total bacteria protein. After purification and cleavage of the fusion protein purified GnRH protein could be obtained. Then the fusion protein was used to immunise some ICR mice to produce anti-GnRH antibody. This fusion protein could significantly elicit specific antibody response in immunized mice compared with the blank groups, and the titers against GnRH reached peak 0.707 +/- 0.320 at the 5th week after immunization. These results demonstrated that recombinant protein could induce high GnRH antibody responses in laboratory animals.
authors.affiliation: Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China.
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Ding
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Cao
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Wu
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D051379:Mice; D008813:Mice, Inbred ICR; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017210:Tilapia / Q000502:physiology*
chemical_list: D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone