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

Collection of Ye Mule Aries sheep ovarian tissue

The ovaries of purebred Ye Mule Aries sheep (healthy ewes that normally produce 3 fetuses) were collected from animals from the pastoral area of Halamumul Township, Emin County, Xinjiang. The ovaries were collected in 1.5 mL centrifuge tubes and placed in a gauze bag. Subsequently, the ovaries were snap-frozen in liquid nitrogen and stored at −80 °C until use.

Extraction of total RNA and the design of synthetic primers

After thoroughly grinding the collected ovarian tissue in liquid nitrogen, total RNA was extracted according to the specification of TRNzol total RNA extraction kit (TIANGEN) and stored at −80 °C. Because eukaryotic genes contain introns, the total RNA of Ye Mule Aries sheep ovary tissue was amplified by RT-PCR to obtain the cDNA fragment of INHα subunit. Primers were designed on the basis on the full-length sequence of INHα (GenBank, XM 004.004955.1). The expected amplified product fragment size was 1,100 bp. The upstream primer, 5′-ATG TGG CTT CAG CTG CTC CTC TTC-3′, and downstream primer, 5-′GAT GCA AGC ACA GTG CTG GGT G-3′, were synthesized by Shanghai Shenggong Bioengineering Co., Ltd.

Reverse transcription, PCR amplification, and ligation of the INHα gene and vector

Reverse transcription was carried out using a reagent kit (Takala) according to the manufacturer’s instructions. The reaction mixture, 20 µL, consisted of 5×Mix (four µL), RNA (two µg), and RNase-free water (16 µL). The reaction was performed at 37 °C for 15 min followed by 85 °C for 5 s; the product was stored at −4 °C.

PCR of the cDNA was performed according to the TaKaRa kit instructions using primers synthesized by Shanghai Biotech Co., Ltd., to obtain the target gene INHα. The reaction mixture, 25 µL, consisted of 2×Mix (12.5 µL), upstream and downstream primers (100 µmol/L, 0.4 µL each), double-distilled water (9.7 µL), and cDNA (two µL, 1.9 ng/µL). After pre-denaturation at 94 °C for 5 min, 35 cycles of 94 °C for 40 s, 68 °C for 40 s, and 72 °C for 1 min 30 s were performed, followed by a final extension at 72 °C for 7 min. The amplified product was subjected to electrophoresis on 1.0% agarose gel.

After the electrophoresis, DNA was recovered and purified according to the instructions of the QuickGel Extraction Kit (Kangwei Co.). Subsequently, the amplified sequence was ligated into the pMD18-T vector at 16 °C overnight. DH5a competent cells were transformed with the ligation product and cultured overnight on LB-coated plates containing 50 µg/mL ampicillin. Five positive clones were selected, and the identity of the recombinant plasmid was initially confirmed by PCR and double enzyme digestion.

Sequencing of the INHα gene and pMD18-T vector

The clones were sequenced by Shanghai Shenggong Bioengineering Co., Ltd. Obtained cDNA sequences were analyzed using DNA-MAN, DNAStar, and Chromas software, as well as by the comparison of the sequence alignment with the Ara cDNA in the GenBank database (accession number XM-004004955).

Construction and characterization of pEGFP-INHα plasmid

The pMD18-INHα plasmid and the eukaryotic expression vector pEGFP-N1 were cut at restriction sites by ScalI and EcoRI enzymes for 4 h at 37 °C. The digested products were subjected to electrophoresis on 1.0% agarose gel, and recovered and purified using a gel recovery kit. The reaction mixture for the construction of recombinant plasmid pEGFP-INHα consisted of the fragment of the pMD18 plasmid containing INHα (six µL, 1.9 ng/µL), eukaryotic expression vector pEGFP-N1 (one µL, 1.9 ng/µL), T4 DNA Ligase (one µL), 10x T4 DNA ligase buffer (two µL), and double-distilled water (10 µL), for a total volume of 20 µL. The ligation reaction was carried out overnight at 16 °C. The ligation product was transformed into E. coli DH5a competent cells, plated on plates containing LB medium with 50 µg/mL kanamycin, and cultured overnight at 37 °C. Five positive clones were randomly selected and the recombinant pEGFP-INHα plasmid was identified by PCR and double enzyme digestion.

Sequencing of the pEGFP-INHα plasmid

The five pEGFP-INHα-positive clones were sequenced by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. Obtained cDNA sequences were analyzed with DNA-MAN, DNAStar, and Mega 5.0 software, and aligned to the INHα subunit gene sequence in the GenBank database.

Transfection of BHK cells and identification of pEGFP-INHα positive cells

The recombinant pEGFP-INHα plasmid was purified and recovered. The plasmid was mixed with Lipofectamine 2000 at a ratio of 4:1 and incubated with BHK cells at 37 °C in the presence of 5% CO₂. After 48 h, over 50% of the cells were positive for green fluorescent protein (GFP), as determined under an inverted epifluorescence microscope. Cells were harvested and divided into two groups, which were subjected to RT-PCR and Western blot analysis, respectively.

Preparation of immunogens and immunization of rabbits

The eukaryotic expression plasmid pEGFP-INHα (optical density ratio OD260/OD280 of 1.8) was mixed with Lipofectamine 2000 to yield the final concentration of one mg/mL. The rabbits to be immunized were injected with 0.2 mL of 0.5% procaine hydrochloride into the muscles on both sides of the leg at 24 and 2 h before the immunization. Each rabbit received an injection of 0.2 mL of the plasmid (one mg/mL) at the same site. After 10 days, the rabbits were immunized again by injection at the same site, but the immunization dose was halved. Control rabbits received an equal amount of physiological saline.

Determination of antibody levels

Antibody levels in the serum of experimental and control rabbits were determined by enzyme-linked immunosorbent assay (ELISA). For this purpose, 100 µL of a coating solution containing 15 µg of the antigen was added to the wells of a plate and incubated overnight at 4 °C. Blood samples were diluted 1:12,800 with 5% skim milk, and 100 µL aliquot was added to the coated wells and incubated at 37 °C for 1 h. After washing, 100 µL of biotinylated antibody diluted 1:1,000 was added and incubated at 37 °C for 1 h. Wells were washed again and 100 µL of streptavidin-horseradish peroxidase (HRP, diluted 1:1,000) was added and incubated at 37 °C for 1 h. After the final washing, color development was carried out by TMB (Sigma Biochemical Co., Ltd.), and the absorbance value was read at 450 nm.

Determination of FSH hormone levels

The levels of FSH in serum obtained from the experimental and control rabbits was determined by radioimmunoassay. The assay kit was purchased from the Northern Institute of Biotechnology. The range of the standard curve was adjusted to be consistent with the range of normal animal hormone levels, as recommended by the manufacturer.

Statistical analysis

Results are expressed as mean ± standard deviation. Data were analyzed by one-way analysis of variance (ANOVA) with Duncan’s Multiple Range test used for pairwise comparisons. All calculations were performed with the SAS 9.2 software (SAS Inst., Cary, North Carolina, USA). Values were considered significantly different at P < 0.05.

Availability of data and materials

The raw data has been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA), and the accession number is KP-113678.1.

Cloning of the Ye Mule Aries sheep INHα gene coding region and construction of recombinant plasmid

Total RNA extracted from the ovary tissue of Ye Mule Aries sheep (absorbance of A260/A280, 1.9) was subjected to agarose gel electrophoresis. Two bands corresponding to the 18S and 28S rRNA were clearly visible (Fig. 1A), indicating that no degradation occurred in the extraction process. Total RNA from the ovary tissue of Ye Mule Aries sheep was reverse transcribed and amplified by PCR using Oligo dT primers. 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 INH α subunit coding region. Subsequently, the product was incorporated into the PMD18-T vector and transformed into E. coli DH5a competent cells. The identity of the cloned gene (KP-113678.1) was confirmed by PCR, restriction enzyme digestion, and sequencing.

In order to express INHα in eukaryotic organisms, the recovered and purified INHα subunit gene was ligated with a linearized eukaryotic expression vector, pEGFP-N1. The ligation product was transfected into E. coli DH5a competent cells, which were cultured in the presence of kanamycin. The formed colonies were verified by PCR and restriction enzyme digestion (Fig. 1C). Moreover, positive colonies were sequenced (Shanghai Shenggong Biological Engineering Technology Service co., LTD); the obtained sequence was consistent with that of the cloned gene, indicating that the pEGFP-INHα eukaryotic expression plasmid was successfully constructed.

Molecular biology analysis of Ye Mule Aries sheep INHα

The cloned expression vector pMD18-INHα was sequenced and detected by Shanghai Shenggong Bioengineering Co., Ltd. The results showed that the full length of the inhibin gene was 1109 bp, in accordance with the target band length, of which 1083 bp constituted the open reading frame (ORF). The ORF consists of the start codon ATG, stop codon TAA, and contains the complete INH α subunit coding region, which codes for 360 amino acid residues. The A, T, G, and C content in the cDNA sequence of the INHα gene was analyzed using DNAMAN software. The percentages of each nucleotide were 13.6%, 20.3%, 30.0%, and 36.1%, respectively. The total G+C content (66.1%) was higher than that of A+T (33.9%). The calculated molecular weight of the protein was 39729 Da. Sequence comparison with the GenBank Ovis aries sequence (accession number: XM_004004955.1) revealed two point mutations (G580A, A636G), giving a nucleotide sequence homology of 96.94% and amino acid homology of 98.90%. Moreover, the nucleotide sequence of the peptide moiety was the same. Additionally, SignalP4.1 software (http://www.cbs.dtu.dk/services/SignalP/) analysis predicted that there was a signal peptide cut point between the 17th and 18th amino acids of the INHα protein (D = 0.861, D-cutoff = 0.450). Thus, the entire protein includes a 17 amino acid signal peptide and a 343 amino acid mature peptide (Figure2A). Tthe NCBI database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) on-line tool Conserved Domain Search Service identified the presence of a transforming growth factor (TGF)-beta domain of at the site from amino acid 253 to 360 of the INHα protein. Moreover, the growth factor TGF-beta domain and one transforming growth factor TGF-ßfamily member active domain were identified at amino acids 256 to 360 (Fig. 2B).

Results obtained using DNAMAN software showed that the length of the amplified INHα gene was 1109bp and the homology to the sequence of human and gorilla genes was 84.4% and 84.3%, respectively. The homology to cattle, horse, goat, sheep, pig, wild boar, dog, cat, rabbit, Mus musculus, brown rat, and rainbow trout genes was 95.2%, 86.1%, 97.9%, 99.8%, 88.1%, 88.4%, 83.6%, 85.5%, 83.4%, 80.4%, 79.0%, and 51.5%, respectively. 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. This data shows that the INHα gene has relatively high homology between different animals, which may indicate that the ability of INHα immunization to increase fecundity is preserved across species (Fig. 2C).

The phylogenetic tree analysis of the Ye Mule Aries sheep INHα gene by Mega5.0 software indicated that this gene follows the expected evolutionary genetics, from aquatic to terrestrial animals, and from low to high organisms. Based on the alignment of the homologues of the INHα subunit gene between the Ye Mule Aries sheep and other animals, the phylogenetic tree clearly reflects the evolutionary genetic characteristics of the organism (Fig. 2D).

Identification of recombinant plasmids in BHK cells by RT-PCR and western blot

The transfection efficiency of the recombinant plasmid (pEGFP-INH α) in BHK cells was analyzed 48 h after transfection on the basis of green fluorescence (Figs. 3A–3C). Moreover, a specific band of 1,109 bp was detected in BHK cells by RT-PCR, which was absent in non-transfected BHK cells (Fig. 1D). The expression of the recombinant plasmid pEGFP-INHα was also confirmed by detection of a specific 40 kDa band in western blot assay. Thus, the pEGFP-INHα plasmid was successfully constructed and expressed in eukaryotic cells (Fig. 3D).

Antibody titer in blood

The level of anti-inhibin antibody in the blood of the immunized rabbits, measured by optical density (OD), gradually increased from day 10 after the first immunization. The initial immunization was followed by the booster immunization after 10 days. All OD measurements were significantly higher in the injected animals than in the control group. These results indicate that antibody production was rapidly stimulated in the rabbits by the primary immunization, and the antibody level in the blood continued to rise after the second immunization, while anti-inhibin antibody was not produced in the control group (Table 1).

Plasma FSH level

After the initial immunization, the levels of FSH in the serum of the immunized group were slightly higher than in the control group. Ten days after the first immunization and 10 days after the second booster immunization, the serum FSH concentration in the immunized rabbits was significantly higher than in the controls. This result indicates that the first immunization with INHα caused a slight increase in serum levels of FSH, which were further significantly increased following the booster immunization (Table 2).