Preliminary Study On DNA Vaccine Immunogenicity And RNA Interference Of Inhibin Alpha Of Ye Mule Aries Sheep

Inhibin（inhibin, INH）, known as follicular inhibin or inhibin A gonad, is a sugar protein hormone secreted from ovarian granulosa cells and sertoli cells, and composed of an alpha subunit and a beta subunit by two disulfide bonds. The inhibin alpha（INH alpha） is physiologically inhibin essential function in vivo. The inhibin is inhibitor of follicle stimulating hormone（FSH） release. For the females, the inhibin can regulate follicle production and increase the number of dominant follicles to mature. For the males, the inhibin mainly promote sperm and testicular spermatogenesis of male. Therefore, animal reproduction could be regulated by inhibiting expression of inhibin A subunit gene, reduce in animal.Inhibin expression in animal can be reduced by two ways. The first way may use antibody against inhibin raised from active or passive immunization to neutralize endogenous inhibin of animal, thereby reducing inhibin level in animal. The second way may use RNA interference to silence the expression of inhibin A subunit, ultimately resulting in reduction of inhibin level in animal. This study used bioinformatics, gene cloning, protein analysis, gene immunization technique to test inhibin alpha subunit gene of Xinjiang Tacheng Ye Mule Aries sheep in the following aspects:1. Cloning and sequence analysis of follicular inhibin alpha of Ye Mule Aries sheepIn order to amplify inhibin A subunit gene, a pair of specific primers were designed according to INH alpha subunit gene sequence from Gen Bank（Gen Bank number: XM₀04004955.1）. Total RNAs were extracted from Emil white sheep ovarian tissue as PCR template. Inhibin A subunit was cloned and analyzed using RT-PCR technology and DNA sequencing. The results showed that the full-length of Mule Aries inhibin gene was 1109 bp, which contains 1083 bp of open reading frame（ORF）（the ATG as start codon, stop codon was TAA, containing the complete inhibin alpha subunit coding sequence）, encoding360 amino acid residues. The inhibin A subunit gene sequence of Ye Mule Aries sheep was submitted to Gen Bank（accession number: KP-113678）.2. Construction of Eukaryotic expression vector of the inhibin alpha subunit geneIn order to construct green fluorescent eukaryotic expressing vector of inhibin alpha subunit gene, we use conventional methods of modern biological technology and Laboratory to connect inhibin alpha subunit gene into the green fluorescent eukaryotic expression vector（p EGFP-N1）, and the green fluorescence of the fusion protein expression vector（p EGFP-INH alpha）, which were confirmed by PCR, double enzyme digestion, sequencing of BHK bacteria liquid, cell transfection and protein expression experiments. The results showed that the green fluorescent eukaryotic expressing vector of inhibin alpha subunit gene（p EGFP-INH） was successfully constructed, and EGFP-INH was expressed and secreted in BHK-21 cells.The transfection rate was as high as 70%. EGFP-INH fusion protein was about 40 KD. The p EGFP-INH vector laid a foundation for developing inhibin gene vaccine.3. Effects of follicular inhibin A subunit gene vaccine on reproductive hormones in rabbitsIn order to study p EGFP-INH alpha mmunogenicity, we use recombinant plasmid p EGFP-INH successfully prepared as immunogen gene to immunize 30 rabbits. ELISA immunoassay（ELISA） was used detect anti inhibin antibody, radioimmunoassay（RIA） was used to measure hormone level in the serum.in different periods after treatment. The results showed that the recombinant plasmid p EGFP-INH induced significantly higher antibody titer than that of control group,（P<0.05）, and antibody levels after the second immunization were significantly increased. After the first immunization, follicle stimulating hormone（FSH）and estradiol（E2） were higher than that in the control group, and the second immunization induced phase difference（P<0.05） before and after the two immunization, but luteinizing hormone（LH） had no obvious change. Our results showed that inhibin gene immunization in rabbits can reduce the content of FSH and inhibin and promote the secretion of E2, which influenced follicle development of animal and animal productive.4. Construction of si RNA expression vector targeting inhibin alpha subunit gene of Ye Mule Aries sheep.In order to construct si RNA expression vector targeting inhibin alpha subunit gene, three different RNAi sites were designed according to the INH gene sequence（Gen Bank:KP-113678.1）. We used RNAi technology and routine laboratory methods to ligated si RNA into the eukaryotic expression vector of RNA interference, and RNAi vector were confirmed by the related detection. The results showed that the plasmid was successfully expressed in sheep granulosa cells, and the transfection rate of around 50%. The si RNA silence rate reached to 83% as showed by Q-PCR and protein analysis experiment. Our results will lay the foundation for developing transgenic cell model of Mule Aries INH gene interference.