Abstract
BackgroundYe 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.MethodsPrimers 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.ResultsThe 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.ConclusionsThe 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.
Highlights
Ye Mule Aries sheep is one of the most important sheep breeds in Xinjiang, China
The obtained product was subjected to agarose gel electrophoresis and yielded a specific band corresponding to the length of 1,109 bp (Fig. 1B), which was preliminarily identified as the Ye Mule Aries sheep inhibin subunit α (INH α) subunit coding region
The comparison of the Ye Mule Aries sheep follicle inhibin gene with other animals revealed that Ye Mule Aries sheep gene had the highest homology with sheep, followed by goat, cattle, wild boar, pig, horse, cat, human, large orangutan, dog, rabbit, Mus musculus, brown rat and rainbow trout genes
Summary
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. Several studies demonstrated that female animals immunized against INH enter the puberty earlier, show an increase in ovulation rate, and display improved fecundity (Medan et al, 2004; Ishigame et al, 2004). Both active and passive immunization with INH increase ovulation rate and lambing in sheep(Kusina et al, 1995; Fray, Wrathall & Knight, 1994; Henderson et al, 1984) and have a similar effect on ovulation and litter size in pigs and cattle (Wheaton et al, 1998; Morris et al, 1993). Based on studies in mice, poultry, pigs, cattle, sheep and other animals, INH immunization has the potential to improve animal fecundity, which is of great significance in both scientific research and agricultural production
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