3 of Newbornsheep lambs from GanSu Agricultural-Farm Ranch Co, Ltd，which provided
the testicular parenchyma was used forthe preparation of primary sheep testicular cells.
Virus and yeast strains and plasmids
ORFV strain (ORFV/QH01/2010) was isolated from the scar of a clinically ORFV infected sheep from Qinghai, was saved in the laboratory of Lanzhou Veterinary Research Institute (LVRI),China.Yeasts strains (NMY51), pBT3-N,pBT3-STE,PPRN-3, pTSu2-APP, pNubG-Fe65 plasmids （Table 1）for YTH experiments were obtained from Shanghai OE Biotech. Co. Ltd (Shanghai,China). 293T cell, pcDNA3.1-3 x HA-N, and pcDNA3.1-3× Flag-N plasmids（Table 1）are stocks in our lab. Cells were cultured using RPMI 1640 (Gibco, Grand Island, New York, USA)supplemented with 10% fetal calf serum (Gibco, Grand Island, New York, USA) and 100 mg/ml penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37 °C.
Construction of YTHcDNA library of sheep testicular cells
All trials were constructed with the approval of the Ethical Committee of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.Neonatal sheep were fully anesthetized with isoflurane inhalation anesthetic administered by face mask (1 to 2%isoflurane) followed by exsanguination as an adjunctive method of euthanasia. These animals were placed indorsal recumbency on the surgical table for placement of vascular introducer sheaths.Preparation of primary neonatal sheep testicular cells was performed as previously described  with few modifications. The testicular parenchyma by ophthalmic tweezers and scissors under sterile conditions. Cut the testicular parenchyma into tissue pieces of 1-2 mm size, and the small tissue pieces were then put into D-hanks solution and pipetted gently several times. After standing for 5-10 min, the supernatant was removed, and the pellet was allowed to digest with lysis buffer containing 0.1% IV collagenase (GIBCO) and 0.25% trypsin (GIBCO) at 4°C for12h.
An equal volume of DMEM medium (HyClone) containing 10% fetal calf serum (GIBCO) was then added to stop the enzymatic digestions. After gentle pipetting several times using a Pasteur pipette, the tissue lysate was filtered with a 200-mesh copper wire screen. The digested solution was then collected and centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and the pellet was washed twice with serum-free culture medium. The DMEM medium containing 10% fetal bovine serum was added to resuspend the cells. The cell viability was to determine by trypan blue staining. Then, the cells at a density of 1-2 × 105 cells/mL placed in a 25 mm cell culture flask and were incubated at 37 °C in a humidified incubator with 5% CO2. After incubation, the culture media containing non-adherent cells were discarded. The DMEM culture medium containing 10%FBS was added to continue purifying the sheep testicular cells and subculture the cells for 5 passages. Finally, the cultural sheep testicular cells were collected and sent to Shanghai OE Biotech. Co. Ltd (Shanghai, China) for construction of the YTH cDNA library. The total RNA of sheep testicular cells was extracted and reverse transcribed into 1st strand cDNA. Following the normalization treatment and short fragment removal, the cDNA of sheep testicular cells was cloned into PPRN-3 vectors (Prey Plasmid).
Bait plasmid construction
Previous,the ORF047 gene has been cloned from the DNA of ORFV, and the recombinant plasmid ofpGEM- ORF047 was the stock in our laboratory.Based onthe characteristics of the bait vector, the specific primers (restriction site underlined): ORF047-F (5′-ATTAACAAG GCCATTACGGCCGGGGCCGCCGCCAGCATCCAGACCACC-3′),ORF047-R (5′- GACGGACGGCGGAAATTCCGTAAAGGGGCCGCCTCGGCCAATCAGTT-3′) was designed. Subsequently, the PCR product was cloned and digestedwith restriction enzymeSfiI (NEB, USA) and inserted intoa pBT3-Nplasmid. The recombinant pBT3-N-ORF047plasmid (bait plasmid)was confirmed by restriction enzyme digestion and sequencing (Sangon Biotech,Shanghai, China).
Bait plasmid expression in yeast cells
Following the manufacture's protocols of the Yeast maker™ Yeast Transformation System 2 kit (Cat. No.630439, Clontech, USA),the transformants of pSTU2-APP plasmids were used as a positive control. ThepPR3-N plasmid was used as a negative control. The pBT3-N-ORF047,pSTU2-APP, pPR3-N plasmids were transformed into NMY51, respectively. Transformants were grown on SD/-Leu, SD/-Trp agar plates at 30°C for 3–5 d. To check the ORF047 bait plasmid expression in NMY51, one colony from the SD/-Trp plate was inoculated into SD/-Trp broth and grown to 0.6 OD600 at 30°C, 250 rpm. The total proteins were subsequently extracted from the centrifuged pelleted cells by the Y-PER yeast protein extraction reagent(Thermo,RF-236781). The extracted proteins were separated by 12% SDS-PAGE and electro-blotted onto PVDF membrane (Millipore, Billerica, Massachusetts, USA) for western blot analysis. The ORF047bait expressionwas detected with anti-Lex A mouse McAb (Cat. No.SC-390386, SantaCruz, USA),followed by m-IgGk BP-HRP second antibody (Cat. No.sc-516102 SantaCruz, USA), with positive signals revealed, all the membranes were imaged using the ChemiDoc XRS + system (Bio-Rad).
The dualmembrane function assay of bait plasmid
Following the manufacture's protocols of the YTH System (Clontech, Mountain View, California,USA), the pBT3-N-ORF047 and pOst1-NubI, pBT3-N-ORF047 and pPR3-N,pSTU2-APP and pNubG-Fe65,pSTU2-APP and pPR3-N plasmids were transformed into NMY51,respectively. Transformants were grown on SD/-Trp-Leu, SD/-Trp-Leu-His, SD/-Trp-Leu-His-Ade agar plates at 30 °C for 4 d.Count the number of colonies on all plates(SD/-Trp-Leu-His and SD/-Trp-Leu-His-Ade) versus nonselective plates SD/-Trp-Leu. Suppose pBT3-N-ORF047 bait is properly expressed and functional in the DUALmembrane functional assay. In that case, we should observe between 10% and 100% growth on SD/-Trp-leu-his and SD/-Trp-leu-his-ade plates derived from transformation reaction of pBT3-N-ORF047 and pOst1-NubI plasmids, depending on the expression level of your baitgrowth under selection indicates that your bait is well expressed and that the Cub moiety is accessible for interaction with the Ost1-NubI moiety expressed from the pOst-NubI control prey. It should observe no significant growth on selective plates derived from the transformation reaction ofpBT3-N-ORF047 and pPR3-N plasmids,as bait does not interact strongly with the NubG fused nonsense-peptide expressed from the pPR3-N control prey. Transformation reaction of pSTU2-APP and pNubG-Fe65 should yield robust growth under selection since the APP bait (pSTU2-APP) is well expressed and interacts strongly with Fe65 prey fusion. Transformation reaction of pSTU2-APP and pPR3-N（the negative control）should yield considerably fewer colonies than a reaction of pSTU2-APP and pNubG-Fe65. If the bait interacts with the Ost1-NubI control preybutnot with the pPR3-N derived NubG-nonsense-peptide prey,the bait plasmid could be used inthe YTH screening.
YTH screeningby co-transformation of bait with prey plasmids
To screen the host proteins tointeract with ORFV-047 bait against a NubG-fused cDNA library ofsheep testicular cells, pBT3-N-ORF047 and prey plasmids were co-transformed into NMY51 with Yeastmaker™ YeastTransformation System according to theDUALmembrane starter kits User Manual(Dualsystems biotech). ThepBT3-N-ORF047 and prey plasmids were co-transformed into NMY51, and the transformants were then grown on SD/-Trp-Leu, SD/-Trp-Leu-His, SD/-Trp-Leu-His-Ade/X-Gal agar plates at 30°C for 3–5 d.Blue colonies were patched out onto higher stringency SD/-Trp-Leu-His-Ade/X-Gal agar plates.Primers ofpPR3-N-F/R wereapplied to amplify each insert DNAof potential positive prey plasmids.
Confirmation of the interactions
pBT3-N-ORF047bait wasco-transformed into NMY51 with each prey plasmid in putatively positive hits to confirm the interactions. The prey plasmids were brieflyextracted from putatively positive clones using the Easy Yeast Plasmid Isolation Kit (Cat. No. 630467, Clontech, Mountain View, California,USA). Subsequently, each prey plasmid was transformedinto E. coli DH5α competent cells (Transgen,Beijing,China), and purified from transformants growing on selected LB/Amp agar plates using the Plasmid MiniKit I (Cat. No. D6943-02, Omega, Doraville, Georgia,USA). Following this, each putatively positive prey plasmid was co-transformed with pBT3-N-ORF047 bait and pBT3-N plasmids into NMY51 and the co-transformants grown on SD/-Trp-Leu-His-Ade/X-Gal plates to test for interactions. Co-transformant containing pSTU2-APP and pNubG-Fe65, grown on SD/-Trp-Leu-His-Ade /X-Gal, was used as a positive control,and co-transformantscontaining pSTU2-APP and pPR3-N, grown on SD/-Trp-Leu-His-Ade /X-Gal, was used as a negative control. Blue colonies indicated true positive interactions under these conditions. To verify positive clones, the prey plasmids were sequenced using the pBT3-N primers, and the sequencing results were analyzed by blasted in NCBI.
Positive prey analysis
The sequencing results were analyzed by blasted in NCBI, which revealed that three inserts had a100% sequence identity to that of the three Ovisaries genes:databases to analyze the corresponding function.
Construction of PABPC1,SERP1, FLC, ORF047 expression plasmid
The expression primers were designed based on the published sequences:SERP1 gene（XP_014948090.1）,the forwardprimer,5′AAGCTTATGGTCGCCAAGCAGCGGA 3′(the underlined sequenceis the HindIII site) and the reverseprimer,5′AATGAATTCTCACATGCCCATCCTGATAC -3′ (the underlined sequence is the EcoR I site).FLCgene(XP_014961714.1)AAGCTTATGAGCTCCCAGATTCGTCAG(the underlined sequence is the HindIII site)and the reverse primer,5'GAATTCCTAGTCGTGCTTGAGGGT3' (the underlined sequence is the EcoR I site). PABPC1 gene (XM_004001826.3), The forward primer,5'AAGCTTATGAACGCTGCGGCCAGCAGCTAC3' (the underlined sequence is the Hind III site),5'GCGGCCGCCTAAGAGGTAGCAGCAGCAAC3' (the underlined sequence is the Not I site).
Sheep testicular cells were collected and adjusted to 1´107/mL,andthe cells were washed with PBS by centrifugation at 2000 gfor 7 min. Total RNA was extracted from the collected cells with the Catrimox-14TM RNA kit (TaKaRa Corporation, China). cDNA was synthesized at 42℃ using oligo (dT)-adaptor primer and avian myeloblastosis virus (AMV) reverse transcriptase.The complete sequence of PABPC1, SERP1, or FLC was amplified from synthesized cDNA based on the product were analyzed by electrophoresis on a 1% agarose gel. The fragment gene with alength of 201bp,528bp,1983bpseparately was subcloned intoa pCDNA3.1-3× Flag-Nexpression vector (Invitrogen, USA).ORF047 gene was subcloned into a pCDNA3.1-3HA-N expression vector. The mutation-free recombination plasmids were confirmed by sequencing and subsequently transformed into JM109 Escherichia coli cells. Recombinant plasmids were selected by blue-white selection (Takara Biotechnology ).
Western blot and co-immunoprecipitation assay
To detect the interaction between ORF047 bait and prey proteins, HEK293T cells were transfected with pcDNA-3.1-HA-ORF047 and cotransfected with either pcDNA-3.1-3× Flag-FLCor pcDNA-3.1-3× Flag-SERP1pcDNA-3.1-3×
Flag-PABPC4plasmids for the Co-IP assay.Cells were harvested and washed two times with cold phosphate-buffered saline (PBS), then pretreated with 0.5 ml NP-40 lysis buffer (Beyotime, P0013F)witha cocktail of many protease inhibitors (1:1000)centrifuged at 12 000 rpm for 15 min at 4°C, and the supernatants were collected. For immunoprecipitation,0.4ml of cell lysate was incubatedwith 1.5 mg beadscoated with anti- FLAGantibody (Sigma )for≥ 60 min on a rotator at 4°C,then the tubes were placedon the magnet for 1 min and remove the supernatant, andthe beads were washed 3 times in 500µl pre-chilled lysis buffer, andresuspendedin 50µl 1´Laemmli (loading) buffer and boil for 5 min at 95°C,the tubes were then put on the magnet for 1 min, and the supernatant wastransferredto fresh tubes.The proteins wereresolved by electrophoresis on 12% Bis-Tris polyacrylamide gels (Shanghai Sangon Biotech, China) and transferred to polyvinylidene fluoride membranes(Immobilon-P Transfer membranes, Millipore). Membranes were blocked for 12 h at 4°C in 5% (wt/vol) Tris-buffered saline supplemented with 0.1% Tween 20 (TBST)-diluted milk (BSA, Amresco) buffer. Membranes were incubated with primary antibody diluted in 5% (wt/vol) BSA and 1´ TBST at room temperature.The primary antibodies used include anti-HA (66006-1, Proteintech), anti-FLAG (F3165, Sigma-Aldrich).The blotswere detected by the enhanced chemiluminescence detection kit (#1705062, Bio-Rad) after incubation with an appropriate secondary antibody conjugated to horseradish peroxidase. All the membranes were imaged withthe ChemiDoc XRS + system (Bio-Rad).