Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide.
To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine.
Xu
Jinshu
J
The Biopharmaceutical College, China Pharmaceutical University, Nanjing, PR China.
Zhu
Zheng
Z
Duan
Peng
P
Li
Wenjia
W
Zhang
Yin
Y
Wu
Jie
J
Hu
Zhuoyi
Z
Roque
Rouel S
RS
Liu
Jingjing
J
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D018984:Epitopes, T-Lymphocyte; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism; D008459:Measles virus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014764:Viral Proteins / Q000235:genetics
D000906:Antibodies; D018984:Epitopes, T-Lymphocyte; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D007987:Gonadotropin-Releasing Hormone