Cloning expression and immunogenicity analysis of inhibin gene in Ye Mule Aries sheep

Abstract

Background Ye Mule Aries sheep is one of the most important sheep breeds in Xinjiang, China. This breed is well adapted to harsh environmental conditions and displays strong disease resistance, fast growth, and high cold tolerance. To analyze the clonal expression and immunogenicity of the Ye Mule Aries sheep inhibin gene, total RNA was extracted from sheep ovarian tissue and used as a template to generate a eukaryotic expression vector and study inhibin immunogenicity. Methods Primers were designed to amplify the inhibin A gene via polymerase chain reaction and the amplified product was cloned between the ScalI and EcoRI restriction sites of the expression vector pEGFP-N1 to construct a recombinant plasmid, pEGFP-INHα. Following the validation of successful cloning, the pEGFP-INHα plasmid was transfected into BHK cells to verify expression in eukaryotes and subsequently utilized as an antigen in rabbits. Rabbits were tested for anti-inhibin antibodies and serum follicle-stimulating hormone (FSH) concentrations. Results The analysis of the INHα gene sequence revealed that INHα is 1109 bp long and is translated to an approximately 40 KDa protein. Bioinformatics approach indicated that the INHα gene is highly conserved between organisms. Immunization with the eukaryotic expression vector, pEGFP-INHα, which expresses the INHα gene elicited immune response and generatigeneration on of anti-INHα antibody. The antibody had a significant regulatory effect on the serum concentration of FSH in rabbits and led to higher levels of FSH, indicating increased ovary function. Conclusions The present work resulted in a successful construction of eukaryotic expression plasmid pEGFP-INHα and verified the immunogenicity of this highly conserved protein. Further, the expression of pEGFP-INHα was shown to have a significant impact on the secretion of FSH, indicating a potential regulatory role in ovarian function. In conclusion, our current findings can serve as a working model for studying the effect of INHα on the breeding performance of Ye Mule Aries sheep, providing a novel strategy to improve their reproduction rates.