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

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.

In 1932, McCullough established that a factor contained in the aqueous extract of bovine testes exerted 29 negative feedback on follicle-stimulating hormone (FSH) secretion; the factor was named "inhibin" (INH 30 or IB) [1] . INH is a glycoprotein hormone secreted by testicular supporting cells and ovarian granulosa cells. 31 The functional form of INH is a dimer containing two disulfide bonds between αand ß-subunits, as well 32 as glycosylation sites on the base [2] . In mammals, the ß-subunit has two isoforms, A and B. Therefore, INH 33 is expressed in two forms: INHA (aßA) and INHB (aßB) [3] . It has been demonstrated that INH is the main 34 negative feedback regulator of FSH secretion in mammals, suppressing the secretion of FSH in the pituitary 35 gland [4][5][6][7][8] . Moreover, the levels of INH can reflect the amount of follicle growth at the beginning of the 98 ℃ for 40 s, and 72 ℃ for 1 min 30 s were performed, followed by a final extension at 72℃ for 7 min. The 99 amplified product was subjected to electrophoresis on 1.0% agarose gel.

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After the electrophoresis, DNA was recovered and purified according to the instructions of the QuickGel   Figure 1C). Moreover, positive colonies were 216 bp was detected in BHK cells by RT-PCR, which was absent in non-transfected BHK cells ( Figure 1D). 217 The expression of the recombinant plasmid pEGFP-INHα was also confirmed by detection of a specific 218 40kDa band in Western blot assay. Thus, the pEGFP-INHα plasmid was successfully constructed and 219 expressed in eukaryotic cells ( Figure 3D). 220 Antibody titer in blood 221 The level of anti-inhibin antibody in the blood of the immunized rabbits, measured by optical density 222 (OD), gradually increased from day 10 after the first immunization. The initial immunization was followed 223 by the booster immunization after 10 days. All OD measurements were significantly higher in the injected 224 animals than in the control group. These results indicate that antibody production was rapidly stimulated in 225 the rabbits by the primary immunization, and the antibody level in the blood continued to rise after the 226 second immunization, while anti-inhibin antibody was not produced in the control group (Table 1).
227 Plasma FSH level 228 After the initial immunization, the levels of FSH in the serum of the immunized group were slightly 229 higher than in the control group. Ten days after the first immunization and 10 days after the second booster 230 immunization, the serum FSH concentration in the immunized rabbits was significantly higher than in the 231 controls. This result indicates that the first immunization with INHα caused a slight increase in serum levels 232 of FSH, which were further significantly increased following the booster immunization (Table 2).

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In this study, we have successfully constructed the eukaryotic expression plasmid pEGFP-INHα and 247 expressed it in BHK cells, along with the eukaryotic expression vector pEGFP-N1 coding for the enhanced 248 green fluorescent protein (EGFP), which was used to detect the expression of exogenous genes in vitro. 249 The expression vector is more convenient for detecting the expression of the fusion gene [37] . The eukaryotic 250 expression system developed here overcomes certain deficiencies present in prokaryotic expression systems 251 because the expression product retains the natural activity of the original protein. Moreover, the expression 252 product is non-toxic and easy to purify, making this system increasingly attractive [38] .
255 FSH release and the maintenance of normal ovarian function. Proper control of FSH is vital due to its effects 256 on follicular growth, development, luteinization, and regulation. Thus, the function of granulosa cells 257 ultimately determines the luteinization and atresia of follicles [39] . Gene immunization is based on the same 258 basic principles as standard immunologic procedures, and inhibin gene immunization is based on 259 conventional gene immunization and INH [40] . Therefore, the inhibin antigen-encoding gene can be inserted 260 into a eukaryotic vector and transformed into the animal, leading to the synthesis of the inhibin antigen 261 protein by the transcriptional system of host cells and its secretion. These processes trigger the generation 262 of the specific immune response of the host and production of an anti-inhibin antibody to neutralize the 263 follicle inhibin. The resulting interaction between the proteins reduces the levels of inhibin, thereby 264 increasing the ovulation rate and sperm production in animals [41] . Given the regulatory mechanism of the 265 inhibin gene and the negative feedback regulation of FSH, as well as current genetic vaccine methods, such 266 as the imatin DNA vaccine pINH immunization of mice [42] , eukaryotic expression plasmid pcINH active 267 immunization of rats [43] , pCIS recombinant plasmid active immunization of rats [44] , and other 268 immunization methods, the results obtained here indicate that inhibin gene immunity can promote follicular 269 development and increase plasma FSH levels.

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In this study, a pEGFP-INHα plasmid was constructed and adopted a scientific immunization method to 271 actively immunize rabbits using the corresponding adjuvant. Anti-inhibin antibodies could be detected in 272 the blood after the first immunization. However the titer of antibodies remained low until the second 273 immunization, which increased antibodies production and resulted in higher titer of the egg yolk antibody 275 especially using genetic recombination technology which has greatly simplified this process. Nevertheless, 276 for practical applications, some macromolecular proteins will require specific structure analysis and 277 selection of suitable vectors and strains to ensure correct expression and preserve the structural and physical 278 properties of the Inhibin A subunits [46] . This strategy opens a novel avenue for the establishment of more 279 specific monoclonal antibodies and has great potential as a new tool for improving the overall fecundity of 280 animals. . In addition, several experimental approaches proved that inhibin immunization not 286 only increases the rate of ovulation but also increases the number of lambs [48][49] , as well as ewes [50] , mice 287 [51] and rats [52] . Inhibin gene immunization can theoretically reduce the level of inhibin in vivo for an 288 extended time, and its immune effect should be equivalent to or better than conventional immunity [47,49,50] . Manuscript to be reviewed 295 inhibin gene will, most likely, benefit the rapid, healthy, and sustainable development of the sheep industry, 296 and solve the "lambing rate" problem of Xinjiang sheep in the future.

298
The present study showed that the Ye Mule Aries sheep follicle inhibin gene was successfully integrated 299 into a highly efficient and stable INHα eukaryotic expression system yielding a biologically active protein.
300 Moreover, the inhibin gene is a regulator of genes responsible for reproductive function and can be utilized 301 to improve the fertility and production performance of animals via immunization-based reduction of the 302 negative feedback of inhibin on the follicle stimulating hormone, estrogen. 309 They are also grateful to the scientific research teams of Ningxia University and Shihezi University for their 310 concern, help, and support.   Manuscript to be reviewed