CN101906163B - Immunocontraceptive synthetic peptide and immunocontraceptive chimeric peptide and application thereof - Google Patents

Abstract

The present invention relates to a synthetic peptide for immune contraception. The chimeric peptide for immune contraception has an amino acid sequence as shown in SEQ ID: 1 in the sequence listing. The synthetic immunocontraceptive peptide hESP _103-107 has an amino acid sequence as shown in SEQID: 2 in the sequence listing. The synthetic immunocontraceptive peptide hIzumo _216-220 has an amino acid sequence as shown in SEQ ID: 3 in the sequence listing. The present invention also provides chimeric peptide, synthetic peptide hESP _103-107 or synthetic peptide hIzumo _216-220 as immune contraceptive vaccine, and observes their application in contraception. The present invention studies for the first time the chimeric peptide composed of cross-reactive antigens of sperm proteins hESP, hIzumo and tNASP and UU, which can produce strong reversible anti-fertility effect after immunizing mice.

Description

Synthetic peptide and chimeric peptide for immunocontraception and application thereof

【Technical field】

The invention relates to the field of immunology, in particular to a synthetic peptide and chimeric peptide for immunocontraception and applications thereof.

【Background technique】

The principle of contraception is to use scientific methods to prevent and interfere with certain links in the normal conception process, so as to avoid pregnancy and prevent birth. The contraceptive method that adopts at present is a lot, and the prior art that is used for immunocontraception both at home and abroad has: 1. anti-gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) vaccine; 2. anti-ovum zona pellucida (ZP) protein vaccine; ③ anti-human chorionic gonadotropin (hCG) vaccine; ④ anti-sperm surface antigen vaccine. The above contraceptive methods have shortcomings such as endocrine balance and sexual dysfunction, autoimmune diseases, and poor birth control effects.

【Content of invention】

The object of the present invention is to provide a chimeric peptide for immunocontraception aiming at the deficiencies in the prior art.

Another object of the present invention is to provide a synthetic immunocontraceptive peptide hESP _103-107 .

Another object of the present invention is to provide a synthetic immunocontraceptive peptide hIzumo _216-220 .

Another object of the present invention is to provide the use of chimeric peptides, synthetic peptides hESP _103-107 and synthetic peptides Izumo _216-220 .

In order to achieve the above object, the technical solution adopted by the present invention is: a chimeric peptide for immunocontraception, the amino acid sequence of which is shown in SEQ ID: 1 in the sequence listing.

In order to achieve the above-mentioned second purpose, the technical solution adopted by the present invention is: a synthetic immunocontraceptive peptide ESP, the amino acid sequence of which is shown in SEQ ID: 2 in the sequence listing.

The amino acid sequence of the synthetic immunocontraceptive peptide hESP _103-112 is shown as SEQ ID: 4 in the sequence listing.

The amino acid sequence of the synthetic immunocontraceptive peptide hESP _98-112 is shown as SEQ ID: 5 in the sequence listing.

In order to achieve the above-mentioned third purpose, the technical solution adopted by the present invention is: a synthetic immunocontraceptive peptide hIzumo _216-220 , characterized in that its amino acid sequence is shown in SEQ ID: 3 in the sequence listing

The synthetic immunocontraceptive peptide hIzumo _212-221 has an amino acid sequence as shown in SEQ ID: 6 in the sequence listing.

The amino acid sequence of the synthetic peptide hIzumo _212-227 for immunocontraception is shown in SEQ ID: 7 in the sequence listing.

For realizing above-mentioned 4th object, the technical scheme that the present invention takes is:

Chimeric peptide, synthetic peptide hESP _103-107 or synthetic peptide Izumo _216-220 as immunocontraceptive vaccine, in contraceptive application.

It should be noted that the sequence of SEQ ID: 1 is NCAYKTTQANK GGG TGGFTPEIGK GGGTSIERLTETK GGG KGKEATLTKP; the sequence of SEQ ID: 2 is TGGFT; the sequence of SEQ ID: 3 is ATLTK; the sequence of SEQ ID: 4 is TGGFTPEIGK; the sequence of SEQ ID: 5 is TTTFPTGGFTPEIGK; SEQ ID: 6 sequence is KGKEATLTKP; SEQ ID: 7 sequence is KGKEATLTKPMVGPED.

The present invention has the advantage that:

1. Invented a cross-reactive antigen between sperm protein hESP and UU436 protein;

2. Invented the short peptide hESP _103-107 with anti-fertility effect;

3. Invented the cross-reactive antigen ATLTK between human sperm protein hIzumo and UU474 protein;

4. Invented the short peptide hIzumo _212-227 with anti-fertility effect, while UU474 protein has no obvious anti-fertility effect;

5. For the first time, the present invention has studied chimeric peptides composed of cross-reactive antigens of sperm proteins hESP, hIzumo and tNASP and UU, which can produce strong reversible anti-fertility effects after immunizing mice.

【Description of drawings】

Figure 1: Prokaryotic fusion expression of 15 peptides pp1/pp2/pp3/pp4, 15% SDS-PAGE after prokaryotic expression of 15 peptides pp1/pp2/pp3/pp4 recombinant fusion expression vector pTSA18-stv Coomassie brilliant blue staining after separation. Lane M: Protein markers; Lane 1: Whole bacterial lysate before induction; Lanes 2, 3, 4, 5: Whole bacterial pellet lysate after pp1/pp2/pp3/pp4 induction in sequence.

Figure 2: Western blot analysis to identify the cross-reactivity of the fusion expressed pentadeceptide pp1-stv/pp2-stv/pp3-stv/pp4-stv, lane 0: whole bacterial lysate induced by empty vector pTSA18-stv; lanes 1, 2, 3, 4: Whole bacterial lysates after induction of fusion peptides pp1-stv/pp2-stv/pp3-stv/pp4-stv in sequence, loading 2 μl/well, rabbit anti-mESP-P1 polyclonal antibody, 1:8000 ; Swimming lanes 5, 6, 7, 8: whole bacterial lysates after induction of peptides pp1-stv/pp2-stv/pp3-stv/pp4-stv, loading 2 μl/well, rabbit pre-immune serum, 1: 8000.

Figure 3: Prokaryotic expression and purification of rUU436 protein, prokaryotic expression of rUU436 protein, after affinity chromatography purification, the eluate from each part was collected in sections, separated by 15% SDS-PAGE and stained with Coomassie brilliant blue, lane M: protein marker; lane 1: The final eluate is the protein sample; Lanes 2-5: The previous eluate contains more miscellaneous proteins.

Figure 4: Western blot analysis identified the cross-reactivity between rUU436 protein and anti-rmESP-P1, rUU436 protein was separated by 15% SDS-PAGE and transferred to PVDF membrane, and it was immunoreactive with rabbit anti-mESP-P1, as shown by ECL. Swimming lanes 1 and 2: rabbit anti-mESP-P1 polyclonal antibody 1:8000; Swimming lanes 3 and 4: Rabbit preimmune serum; Swimming lanes 2 and 4: rUU436 protein; Swimming lanes 1 and 3: Antigen-free samples.

Figure 5: ELISA detection of anti-synthetic peptide hESP _98-112 and anti-rUU436 antibodies in immunized mouse serum, synthetic peptide hESP _98-112 and rUU436 protein immunized mouse antiserum and pre-immune serum were used as primary antibodies, diluted 1:100, Measure the absorbance.

Figure 6: ELISA detection of anti-peptide antibody titer in rabbit serum after immunization with synthetic peptide hESP _98-112 .

Figure 7A: Western-blot verification of rabbit anti-peptide antibody cross-reactivity, lanes 1, 3: rUU436; lanes 2, 4: rmESP-P1; lanes 1, 2: rabbit anti-peptide antibody, 1:2000; lane 3, 4: Rabbit pre-immune serum, 1:2000.

Figure 7B: Western-blot verification of cross-reactivity of rabbit anti-peptide antibody, lanes 1 and 2: human sperm protein extract; lanes 3 and 4: mouse sperm protein extract; lanes 1 and 3: rabbit anti-peptide Antibody, 1:2000; lanes 2 and 4: rabbit pre-immune serum, 1:2000.

Figure 8: Western-blot verification of rabbit anti-rUU436 antibody cross-reactivity, lanes 1, 4: rUU436; lanes 2, 5: rmESP-P1; lanes 3, 6: mouse sperm protein extract; lanes 1, 2, 3 : mouse anti-rUU436 antibody, 1:2000; lanes 4, 5, 6: mouse preimmune serum, 1:2000.

Figure 9: Verification of common antigen TGGFT as BCE by competitive ELISA.

Figure 10: Detection of rabbit anti-peptide antibody and human sperm immune reaction by indirect immunofluorescence technique (scale bar = 5 μm), A, B, C: serum after rabbit anti-peptide immunization, 1:50; A1, B1, C1: rabbit immunization Pre-serum; FITC-labeled goat anti-rabbit IgG (1:500) detection, laser scanning confocal microscope observation; A, A1: fluorescence images; B, B1: light microscopy images; C, C1: composite images of fluorescence and light microscopy.

Figure 11: Detection of rabbit anti-peptide antibody and mouse sperm immune reaction by indirect immunofluorescence (scale bar = 5 μm), A, B, C: rabbit anti-peptide immunized serum, 1:50; A1, B1, C1: rabbit Pre-immune serum; FITC-labeled goat anti-rabbit IgG (1:500) detection, laser scanning confocal microscope observation; A, A1: fluorescence images; B, B1: light microscopy images; C, C1: composite images of fluorescence and light microscopy .

Figure 12: Indirect immunofluorescence detection of mouse anti-rUU436 antibody and mouse sperm immune reaction (scale bar = 5 μm), A, B, C: serum of mice after immunization with anti-rUU436, 1:50; A1, B1, C1 : Mouse pre-immune serum, 1:50; FITC-labeled goat anti-rabbit IgG (1:500) detection, laser scanning confocal microscope observation; A, A1: fluorescence image; B, B1: light microscope image; C, C1 : Composite image of fluorescence and light microscopy.

Figure 13: Effects of anti-synthetic peptide hESP _98-112 antibody and anti-rUU436 antibody on mouse sperm-egg adhesion in vitro.

Figure 14: Effects of anti-synthetic peptide hESP98-112 antibody and anti-rUU436 antibody on mouse sperm-egg fusion in vitro.

Figure 15: Effect of rabbit anti-synthetic peptide antibody on mouse sperm-egg interaction in vitro, capacitated mouse sperm were pretreated with rabbit anti-synthetic peptide antiserum (1:10), and co-incubated with mouse zona pellucida ova. After staining with Hoechst33342 (10 μg/ml); pressed into pieces, observed under a laser scanning confocal microscope, left: fluorescence image, right: phase contrast image.

Figure 16: Effect of rabbit pre-immune serum on mouse sperm-egg interaction in vitro (control). Capacitated mouse sperm were pretreated with rabbit pre-immune serum (1:10) and co-incubated with mouse zona pellucida ova. After staining with Hoechst33342 (10 μg/ml); pressed into pieces, observed under a laser scanning confocal microscope, left: fluorescence image, right: phase contrast image.

Figure 17: Stv-Izumo (Izumo _212-216 , Izumo _216-220 , Izumo _220-224 ) fusion protein expression results, Stv-Izumo (Izumo _212-216 , Izumo _216-220 , Izumo _220-224 ) fusion protein in BL21 (DE3) was expressed in bacteria, and analyzed by Cowes brilliant blue staining after 15% SDS-PAGE electrophoresis. M: low molecular weight protein standard; 1: Stv-Izumo _220-224 ; 2: Stv-Izumo _216-220 ; 3: Stv-Izumo _212-216 ; 4: Stv-β8.

Figure 18: Western blot analysis of B cell epitopes, T0: Stv-β8 does not react with anti-PB; T1: Stv-Izumo _220-224 has a weak reaction with anti-PB; 3: Stv-Izumo _216-220 reacts with anti -PB has a strong reaction; 4: Stv-Izumo _212-216 has a weak reaction with anti-PB.

Figure 19: PCR amplification results of target gene fragments, M: DNA molecule ladder; 1: UU474 (524bp).

Figure 20: UU474 protein expression and purification results, UU 474 protein was expressed in BL21 (DE3) bacteria, after 15% SDS-PAGE electrophoresis, it was analyzed by Cowes brilliant blue staining. M: low molecular weight protein standard; 1: whole bacteria before induction; 2: whole bacteria after induction; 3: protein purified by Ni2+-NTA His- column.

Figure 21: Scatter diagram of antiserum titer titer, note: the antigen was coated with a 96-well plate, the titer of the antiserum was detected with an Anthos Zenyth 1100 Multimode microplate reader, and the antisera of Izumo _212-227 and UU474 were at A485 The absorbance value was significantly higher than that of the pre-immune serum (control).

Figure 22: Western blot method to verify the cross-reaction between sperm protein Izumo and UU474 protein, 1: anti-Izumo _212-227 IgG can have a strong reaction with PB protein; 2: UU474 protein and anti-Izumo _212-227 IgG has a weaker response.

Figure 23: Western blot verification of cross-reactivity between anti-UU474IgG or anti-Izumo _212-227 IgG and human sperm protein, 1: pre-immune serum is the primary antibody; 2: anti-Izumo _212-227 IgG is the primary antibody; 3: anti -UU474IgG is the primary antibody.

Figure 24: Indirect immunofluorescence localization of human sperm smear, (a) localization of anti-Izumo _212-227 IgG fluorescent staining; (c) natural light state of a; (b) localization of anti-CD46IgG fluorescent staining; (d) localization of a Overlapping state with b (scale bar: 5 μm).

Figure 25: Indirect immunofluorescence localization of human sperm smear, (a) anti-UU474IgG fluorescent staining localization; (c) natural light state of a; (b) anti-CD46IgG fluorescent staining localization; (d) overlap of a and b state (scale bar: 5 μm).

Figure 26: Indirect immunofluorescence localization of human sperm smear (pre-immune serum) (e) localization of fluorescent staining in pre-immune serum; (g) natural light state of a; (f) localization of anti-CD46IgG fluorescent staining; (h) localization of a Overlapping state with b (scale bar: 5 μm).

Figure 27: The results of mouse sperm-egg combination and sperm penetrating experiments. Capacitated mouse sperm were incubated with different concentrations of antiserum IgG and pre-immune serum, and incubated with mouse eggs without zona pellucida; (A ): the sperm-egg fusion experiment of anti-Izumo _212-227 IgG diluted in different concentrations; (B): the sperm-egg fusion experiment of anti-UU474IgG diluted in different concentrations; The fusion rate was observed under a confocal microscope. Bars represent standard errors. (N = number of eggs per group).

Figure 28: ELISA detection of anti-CP1 antibody in mouse serum at different times after immunization, 96-well polystyrene plate was coated with 1 μg/well CP1, and mice were injected from the eyes at 1 week, 5 weeks and 8 weeks after immunization Blood was collected from the canthus vein, and the serum was diluted 1:100 as the primary antibody, and the absorbance value at 450 nm was measured. The pre-immune serum and the serum of the control group were used as negative controls.

Figure 29: ELISA detection of anti-CP1 antibody in rabbit serum after immunization. 1 μg/well CP1 was used to coat a 96-well polystyrene plate, and the serum was diluted multiple times after rabbit immunization to detect the titer of anti-CP1 antibody in rabbit serum.

Figure 30: ELISA detection of epitope antibodies in mouse serum after CP1 immunization, 96-well polystyrene plates were coated with 0.5 μg/well recombinant mouse sperm proteins rmESP-P1, rmtNASP, and rmIzumo, and serum after CP1 immunization was used as the primary antibody , diluted 1:500, measured the absorbance at 450nm, and set the pre-immune serum as the control. The results showed that the sera after CP1 immunization had obvious immune reactions with rmESP-P1 and rmtNASP, but had no positive reaction with rmIzumo.

Figure 31: Western-blot analysis of cross-reaction between rabbit anti-CP1 antibody and original recombinant protein, lanes 1, 4: rmIzumo; lanes 2, 5: rmESP-P1; lanes 3, 6: rmtNASP; lanes 1, 2, 3: rabbit Serum after anti-CP1 immunization, 1:2000; Swimming lanes 4, 5, 6: Rabbit pre-immune serum, 1:2000.

Figure 32: Western-blot analysis of cross-reaction between rabbit anti-CP1 antibody and sperm protein extract, lanes 1 and 3: human sperm protein extract was loaded at 60 μg/well; lanes 2 and 4: mouse sperm protein extract was loaded at 50 μg/well Wells; lanes 1 and 2: rabbit anti-CP1 serum, 1:2000; lanes 2, 4: rabbit serum before immunization, 1:2000.

Figure 33: Effect of rabbit anti-CP1 antibody on mouse sperm-egg adhesion in vitro, Bars represent mean±SEM; numbers above Bars represent the average number of eggs in each group; *p<0.05.

Figure 34: Effect of rabbit anti-CP1 antibody on mouse sperm-egg fusion in vitro, Bars represent mean±SEM; numbers above Bars represent the average number of eggs in each group; *p<0.05.

Figure 35: Effect of rabbit anti-CP1 antibody on mouse sperm-egg interaction in vitro. Capacitated mouse sperm were pretreated with rabbit anti-CP1 antiserum (1:10 dilution), incubated with superovulated mouse eggs, and tested by Hoechst After staining with 33342 (10 μg/ml); pressed into pieces, observed under a laser scanning confocal microscope, left: fluorescence image, right: phase contrast image.

Figure 36: The effect of rabbit pre-immune serum on mouse sperm-egg interaction in vitro (control). Capacitated mouse sperm were pretreated with rabbit pre-immune serum (1:10 dilution) and co-incubated with superovulated mouse eggs. After staining with Hoechst33342 (10 μg/ml); pressed into pieces, observed under a laser scanning confocal microscope, left: fluorescence image, right: phase contrast image.

【Detailed ways】

The specific implementation manners of the present invention will be described in detail below in conjunction with the accompanying drawings.

Example 1

Effects of ESP protein and cross-reactive antigen of Ureaplasma urealyticum on fertility

1. Materials

1.1 Experimental animals and specimens

6-month-old (weight about 2.5kg) female New Zealand white rabbits and BALB/c mice (8-12W) were purchased from the Department of Animal Science, Shanghai Jiaotong University School of Medicine, from the Experimental Animal Center of the Chinese Academy of Sciences, and normal human semen samples were obtained from Shanghai Jiaotong University Medicine Provided by the human sperm bank clinic of Renji Hospital Affiliated to the hospital.

1.2 Main reagents

Oligonucleotide sequences and peptides were synthesized by Shanghai Sangon Biotechnology Company, and keyhole limpet hemocyanin (KLH) was purchased from Shanghai Sangon Biotechnology Company. Plasmid pTSA18 was purchased from Shanghai Institute of Family Planning Science and Technology.

2.2.1.3 Experimental instruments Mini Protean 3 system SDS-PAGE electrophoresis instrument and MiniSemi-Dry Trans Blot transfer membrane instrument (Bio-Rad), LSM-510 laser confocal microscope ((CarlZeiss LSM-510, Jena, Germany); Centrifuge 5415R Low-temperature centrifuge (Eppendorf); CO ₂ incubator (SANYO); automatic semen analyzer (Hamilton-Thorn).

Second, the method

2.1 Western blot identification of cross-reactive antigens between ESP _7-242 and UU

1. Chemical synthesis of single-stranded oligonucleotides

The 15-peptide (including the common pentapeptide, underlined) coding oligonucleotide sequence in the chemically synthesized hESP protein has 1OD of sense strand and antisense strand. pp1: QVL ENLVR SVPSGEP (aa38-52)

pp2: STE NDVLT NPISEET (aa84-98)

pp3: TTTFP TGGFT PEIGK (aa98-112)

pp4: N VSIVL HAEEPYIEN (aa128-142)

Add SalI and BamHI restriction site cohesive ends to both ends respectively.

2. Annealing

Take 10 μl of the sense strand and the antisense strand each, at a final concentration of 5 μM, denature at 95°C for 5 minutes, and naturally return to room temperature for annealing.

3. Construction of recombinant pentapeptide fusion expression vector

The annealed product was connected to the vector pTSA18-stv which had been digested with double enzymes. The reaction system is as follows: pTSA18-stv 1 μl, annealed product 5 μl, Ligation Solution Buffer 1.5 μl, T ₄ DNA ligase 1 μl, dd H ₂ O 6.5 μl. The total reaction volume was 15 μl. Incubate at 16°C for 8h.

Take 5 μl of the ligation product to transform E.coli DH5a competent bacteria, and take 100 μl of the transformation product to spread evenly on the LB solid medium plate containing Amp. Incubate at 37°C for 12-14 hours. Pick a single colony into 5ml LB medium (containing Amp). The test tube was shaken overnight at 250 rpm at 37°C in a shaker. Take 1.5ml of the overnight bacteria and use the plasmid mini-extraction kit to extract the plasmid to obtain the recombinant fusion expression vector.

4. Induced Fusion Expression of Pentacapeptide

Take 1 μl small sample of plasmid to transform BL21(DE3) competent bacteria, and spread the transformation product evenly on the LB solid medium plate containing Amp. Incubate at 37°C for 12-14 hours.

Pick out the monoclonal colony to the test tube that has added 5ml LB medium (containing Amp). The test tube was shaken overnight at 250 rpm at 37°C in a shaker. Inoculate 100μ of the overnight bacteria into a 5ml LB medium (containing Amp) test tube, shake the bacteria at 280rpm and 37°C until the OD value is 0.6-0.8, and take 1ml of the bacteria solution from the ultra-clean bench as a negative control. Add 1mM IPTG and continue shaking at 37°C for 4h to induce expression. Set empty plasmid as control.

Take 1ml of the induced bacterial solution and the pre-induced bacterial solution and centrifuge at 6000g for 5min, drain the medium to obtain the precipitate, that is, the bacterial cell, add 400μl ddH ₂ O to resuspend the bacterial pellet, take 20μl of the whole bacterial protein sample and add 5μl of 5× denatured protein loading buffer , denatured at 95°C for 10 minutes; 15% SDS-PAGE, 120V constant voltage electrophoresis for about 2 hours, until the dye reached the bottom of the gel.

Stain with Coomassie Brilliant Blue R-250, decolorize with decolorization solution until the bands are clear, and observe the induced expression results.

5.Western blot analysis of cross-reactivity between fusion expression pentapeptide and anti-mESP-P1 antibody

Take 2 μl whole bacterial protein sample for 15% SDS-PAGE, transfer to PVDF membrane according to the above experimental method, and carry out Western blot analysis. The anti-mESP protein recombinant fragment P1 antibody (anti-mESP-P1) was used as the primary antibody, and the expression product induced by the empty plasmid and the whole bacterial protein before induction were used as negative controls. The cross-reactivity of the anti-mESP-P1 antibody with the above-mentioned 15-peptide was detected.

2.2 Synthesis of short peptides

The fifteen peptide TTTFPTGGFTPEIGK (hESP _98-112 ) was synthesized by Shanghai Sangon Biotechnology Company. Partially coupled KLH was used as antigen to immunize animals, and a small amount of uncoupled KLH was used as antigen to detect antibody titer by EILSA.

2.3 Cloning and expression of rUU436 protein

Serum-8 type UU culture product was taken, and its total DNA was obtained by boiling with water (95°C, 10 min) as a template. Since TGGFT is located at aa90-94 of UU436 protein, the truncated protein fragment clone expresses aa78-246 (UU436 _78-246 ), PCR amplification primers containing TGGFT, UU436 _78-246 sense: 5'GC- GGATCC attaaaaattttgttatagctgat3', antisense: 5 'GC- CTCGAG ttcatgcttttgtaaaaatatcat 3', the target fragment is 507bp in length. GGATCC is the BamH I restriction site, and CTCGAG is the Xho I restriction site. The recombinant expression vector pET-28a(+) with His tag was cloned, constructed and amplified. The recombinant expression vector pET-28a(+) was transformed into E.coli BL21(DE3), and 15% SDS-PAGE electrophoresis analysis confirmed whether the target protein was expressed. The cross-reactivity of rUU436 _78-246 protein fragment with anti-mESP-P1 was analyzed by Western blot.

Experimental steps for mass expression of rUU436 _78-246 protein and Ni ²⁺ -NTA-His affinity chromatography purification:

Purify the expressed recombinant target protein according to the instructions of the Ni-NTA column affinity chromatography kit. The bacterial liquid was centrifuged at 10000rpm×10min, and the bacterial pellet was collected. Resuspend the pellet with double distilled water, sonicate the bacteria on ice, centrifuge again at 10000rpm×10min, and discard the supernatant. Add 5ml of Lysis Buffer B to each gram of the pellet, resuspend the pellet in Buffer B for lysis, and aspirate the supernatant for later use. The supernatant of the lysate was passed through a Ni-NTA column, and the filtrate was discarded. Wash Buffer C to elute the impurity protein in the column, 4ml×3 times, collect the effluent each time. Elution Buffer D elutes the target protein in the column, 1.5ml×4 times, and collects the effluent each time. BufferE elutes the target protein in the column, 1.5ml×4 times, and collects the effluent each time. Equilibrate the column with 10ml Buffer B, store the column at 4°C, and it can be used repeatedly. 15% SDS-PAGE was used to identify whether the target protein was contained in the effluent.

2.4 Preparation of antiserum

The synthetic peptide hESP _98-112 coupled with KLH was used as the immunogen to immunize New Zealand female white rabbits. According to the above-mentioned experimental method, the immunization was carried out three times, and the amount of antigen in each inoculation was about 0.8 mg. On the 35th day after the first inoculation, the post-immunization serum was obtained by bleeding from the carotid artery.

2.5 Mouse mating experiment

BALB/c female and male mice (10-12W) that had undergone fertility verification were used in this experiment, and were randomly divided into groups. KLH-coupled synthetic peptide hESP _98-112 and purified rUU436 protein were used as immunogens to immunize female BALB/c mice. The amount of antigen in each inoculation was about 100 μg, a total of three times. See the first chapter for specific steps. On the 35th day after the first inoculation, blood was collected from the inner canthus vein of the eye to obtain post-immunization serum, and they were mated in cages with a male-to-female ratio of 2:1, and caged for one week. The weight of the female mice was regularly weighed to monitor the pregnancy status, and the litter size of the pregnant mice was counted.

2.6 Competitive ELISA

Referring to other experiments, the anti-mESP-P1 antibody was diluted 1:1000 and incubated with different concentrations (0.1-1 mM) of the synthetic peptide hESP _98-112 at 37°C for 1 hour. The absorbed antibody was used as the primary antibody to react with the 96-well polystyrene plate pre-coated with recombinant mESP-P1, and the ELISA experiment procedure was followed. Another two groups of controls were set up: a group of anti-mESP-P1 antibodies with the same dilution but untreated as positive controls; another group of anti-mESP-P1 antibodies with different concentrations (0.1-1mM) of recombinant mESP-P1 fragments After incubation and absorption, ELISA was performed as the primary antibody, and the absorbance value was measured. The percentage of residual antibody that was not absorbed was calculated.

2.7Western blot analysis

Recombinant rUU436 protein and mESP-P1 fragments and sperm whole protein extracts were subjected to SDS-PAGE, using anti-synthetic peptide hESP _98-112 (anti-peptide) serum and anti-rUU436 (anti-rUU436) serum as primary antibodies, according to the aforementioned method Western blot to analyze the specificity and cross-reactivity of anti-peptide antibody and anti-rUU436 antibody.

2.8 Indirect immunofluorescent staining of human and mouse sperm smears

Normal human semen samples were liquefied at 37°C for 30 minutes, then centrifuged (1000g, 15 minutes) to discard the seminal plasma. Sperm was separated by centrifugation on the upper layer of 45% Percoll, 1000g, 15min. Finally, resuspend and wash twice with PBS for making sperm smears. The method for making a sperm smear of the tail of the mouse epididymis is as follows: Sperm preparation after the acrosome reaction: 10-12 week-old BALB/c male mice were killed by cervical dislocation, and the tail of the epididymis was taken out and placed in preheated M16 (containing 3% BSA) , cut the tissue slightly with surgical scissors, and let it stand in a 5% CO ₂ incubator at 37°C for 30 minutes (sperm will automatically swim out from the tail tissue of the epididymis), then add A23187 (5 μM) and continue at 37°C, Incubate for 1.5 h in a 5% CO ₂ incubator to induce acrosome reaction. Smear the above-mentioned different sperm, that is, the epididymal tail sperm and the acrosome-reacted sperm, and dry them at room temperature. Anti-peptide antibody and anti-rUU436 antibody were used as the primary antibody, diluted 1:50, and FITC-labeled goat anti-rabbit IgG was used as the secondary antibody for indirect immunofluorescent staining of human and mouse sperm, and the serum before immunization was used as the negative control. Method: Pre-cooled acetone fixation for 15min. Rinse gently with PBS. Block with PBS containing 5% BSA for 1 h at room temperature. Anti-P1/P2/P3 rabbit serum was used as the primary antibody, diluted 1:500, and incubated overnight at 4°C. Rabbit pre-immune serum was used as negative control, and the sample without primary antibody was used as blank control. Wash gently with PBS, use FITC-labeled goat anti-rabbit IgG as the secondary antibody, dilute 1:500, and incubate at 37°C for 1h. After gentle washing, the slides were mounted with PBS and observed under a laser scanning confocal microscope.

2.9 Mouse sperm in vitro penetrating experiment

Experimental steps of mouse superovulation, gamete preparation and in vitro piercing

1. Egg retrieval

BALB/c female mice aged 8 to 10 weeks were intraperitoneally injected with 10 IU of pregnant horse serum (PMSG), 48 hours later injected with the same dose of hCG, and 15 to 16 hours later, eggs were collected from the ampulla of oviduct.

2. Sperm collection and antibody processing

10-12 weeks old BALB/c male mice were killed by cervical dislocation, the tail of the epididymis was taken out, placed in preheated M16 (containing 3% BSA), the epididymis tissue was slightly cut with surgical scissors, and placed at 37°C, 5% CO _2Stay in the incubator for 10min, prepare the sperm suspension by the upstream method, adjust the sperm density to 1.0×10 ⁶ /ml, incubate at 37°C, 5% CO ₂ incubator for 1h, add Ca ²⁺ to induce A23187 solution, make The final concentration was 5 μmol/L, and the incubation was continued for 20 minutes. Anti-P1, anti-P2, anti-P3 antiserum and pre-immune serum with different dilution concentrations were incubated with capacitated sperm for 30min. Serum was used to inactivate complement at 56°C for 30 minutes before use.

3. In vitro penetration experiment of mouse sperm

After co-incubating zona pellucida egg cells and differently treated sperm suspensions at 37°C in a 5% CO ₂ incubator for 3-4 hours, aspirate the egg cells, remove the sperm loosely attached to the egg cells, fix with 2% paraformaldehyde for 20 min, After washing with PBS, stain with Hoechst 33342 (10 μg/ml) at 37°C for 15-20 minutes; after washing with PBS, put them on slides and observe with a laser scanning confocal microscope. The number of sperm adhering to the egg membrane was counted under an inverted phase-contrast microscope, and the fusion of sperm and eggs was observed under a fluorescent microscope. The criterion for judging the fusion of sperm and eggs was: the swollen sperm head or pronucleus was seen in the egg cell. The experiment was repeated 3 times.

Anti-synthetic peptide serum and anti-rUU436 serum were inactivated at 56°C for 30 minutes to complement, and incubated with sperm for 30 minutes at different dilutions of 1:10, 1:20, and 1:100. After the treatment, the spermatozoa were co-incubated with the zona pellucida mouse eggs at 37° C., 5% CO ₂ , for 3-4 hours. Sperm loosely attached to the egg cells were removed, fixed with 2% paraformaldehyde for 20 min, washed with PBS and stained with Hoechst 33342 (10 μg/ml) for 15-20 min; washed with PBS, placed on slides and observed under a laser scanning confocal microscope. The number of sperm adhering to the egg membrane was counted under an inverted phase-contrast microscope, and the fusion of sperm and eggs was observed under a fluorescent microscope. The criterion for judging the fusion of sperm and eggs was: the swollen sperm head or pronucleus was seen in the egg cell. The experiment was repeated 3 times.

2.10 Statistical Analysis

The in vitro experiments were repeated more than three times, and the results of each group were expressed as mean ± standard error (mean ± SEM). The litter size of mice mating experiments was expressed as mean ± standard deviation (mean ± SD). The difference between the experimental group and the control group was analyzed by group t-test, using SAS6.12 software for statistical analysis, and p<0.05 was considered statistically significant.

Three, the result

The Common Pentapeptide of 1hESP and UU Protein and the Sequence Homology Comparison with Mouse ESP (mESP)

Bioinformatics technology searched the Swiss-prot database, and found that the N-terminal (aa7-243) of the hESP protein had five common pentapeptides with the UU protein, and had varying degrees of homology with the mESP sequence at the corresponding position. hESP protein aa41-45 shares common pentapeptide ENLVR with UU488aa135-139; corresponds to QNLIM of mESP, 40% identity, 20% similarity. aa87-91 of hESP shares common pentapeptide NDVLT with UU019aa180-184; corresponds to SDVLI of mESP, 60% identity, 20% similarity. aa103-107 of hESP and UU436aa90-94 share a common pentapeptide TGGFT; corresponding to TRGFT of mESP, 80% identity. hESP's aa123-127 and UU301aa79-83 have a common pentapeptide SIKPN. Human CASC5 protein (Cancer susceptibility candidate gene 5protein) also has pentapeptide SIKPN (aa1582-1586), mESP is SIRPN, 80% identity, 20% similarity. CASC5 is highly expressed in sperm cells in the testis and can also be detected in other tissues, so it is not suitable for immunocontraceptive research. hESPaa129-133 has a common pentapeptide VSIVL with UU513aa44-48; corresponds to ISVVL of mESP, 60% identity, 40% similarity. Human PKDREJ protein (Polycystic kidney disease and receptor for egg jelly-related protein) also has the pentapeptide VSIVL (aa1077-1081). PKDREJ protein is only expressed in the testis, and may play an important role in the fertilization process by forming calcium ion channels to initiate the acrosome reaction.

2 Identification of cross-reactive antigens

Chemically synthesize the oligonucleotide chains of pentapeptides PP1 (including ENLVR), PP2 (including NDVLT), PP3 (including TGGFT), and PP4 (including VSIVL) that contain common pentapeptides, and form double strands after denaturation and annealing. The cohesive end of the cleavage site was connected with the vector pTSA18 to construct a recombinant expression vector. After clonal amplification and IPTG induction, the fusion expression of pentadeceptide and streptavidin (stv) was performed. SDS-PAGE analysis of the whole bacterial protein before and after induction showed a new band near 11kDa (Figure 1). The antibody against the P1 peptide of recombinant mESP (anti-rmESP-P1) was used as the primary antibody, and the fusion-expressed 15-peptide was used as the antigen, and the cross-reactivity between the two was analyzed by Western blot. The results showed that PP3 had the strongest immune reaction with anti-rmESP-P1 antibody, and PP3 contained the common pentapeptide TGGFT between aa103-107 of hESP and aa90-94 of UU436; suggesting that the common pentapeptide TGGFT was a cross-reactive antigen (Figure 2).

3UU436 protein clone expression

SDS-PAGE analysis of UU436 protein expression showed a molecular weight of 19kDa. Since the recombinant protein had a His tag, the actual molecular weight was slightly larger than the theoretical molecular weight. Compared with the bacteria before induction, the whole bacterial lysate after induction had a protein expression band at the expected position, but the expression level was not high enough, and the target protein was obtained by affinity chromatography purification (Figure 3). Western blot analysis of the cross-reactivity of the anti-rmESP-P1 antibody with the rUU436 protein showed that the rUU436 fragment containing TGGFT had an immunoreactive band, and the induced prebacterial total protein and the pre-immune serum control had no immunoreactive band (Figure 4). This result indicated that rUU436 protein could cross-react with anti-rmESP-P1 antibody.

4 Detection of serum antibody titer after immunization with synthetic pentapeptide hESP _98-112 and rUU436 protein

To synthesize the pentapeptide hESP _98-112 by chemical method, the hydrophilicity, antigenicity and surface accessibility of the synthesized peptide should be predicted first. Therefore, we used DNAstar analysis software to analyze the N-terminal of hESP, which showed that hESP _98-112 has good hydrophilicity, antigenicity and surface accessibility. The synthetic peptide was coupled with KLH to enhance the immunogenicity, and the titer of the serum antibody obtained after immunizing rabbits and mice was determined by ELISA. Serum before immunization was used as a control, and 96-well ELISA plates were coated with rUU436 and uncoupled KLH-uncoupled pentacapeptide hESP _98-112 as antigens. After immunization, both mouse and rabbit serum produced specific antibodies, and the antibody titer of rabbit serum was about 1:10000 (Fig. 5, 6).

5 Cross-reactivity of anti-synthetic peptide hESP _98-112 antibody and anti-rUU436 antibody

Anti-synthetic peptide hESP _98-112 rabbit serum was used as the primary antibody, and rmESP-P1, human and mouse sperm protein extracts were used as antigens for Western blot analysis. There was a positive reaction band at the position of the target protein, rmESP-P1, The molecular weights of rUU436 are about 25kDa and 24kDa, respectively, and the theoretical molecular weights of hESP and mESP are 38kDa and 45kDa, respectively (Figure 7A, Figure 7B). It shows that the antibody produced by the synthetic peptide hESP _98-112 has specificity and can recognize the original recombinant and natural protein mESP.

Anti-rUU436 serum was used as the primary antibody, and rUU436, rmESP-P1 and mouse sperm protein extracts were used as antigens for Western blot analysis. There was a positive reaction band at the position of the target protein, and the molecular weights of rmESP-P1 and rUU436 were about 25kDa , 24kDa, mESP theoretical molecular weight is 45kDa (Figure 8). The results indicated that the anti-rUU436 antibody could recognize rUU436 protein and also had cross-reactivity with mESP.

6 cross-reactive antigens TGGFT for BCE

In order to further verify whether the cross-reactive antigen TGGFT of UU436 protein and hESP protein is BCE, rmESP-P1 was used as the antigen to coat the ELISA plate, and different concentrations of synthetic peptides were incubated with anti-rmESP-P1 and absorbed as the primary antibody. Another group was incubated with the same concentration of rmESP-P1 to absorb antibodies as a control. The ratio of the measured absorbance value to the absorbance value of the unabsorbed antibody immune reaction was used to calculate the percentage of residual antibody ( FIG. 9 ). Competitive ELISA showed that the synthetic peptide could partially neutralize the anti-rmESP-P1 antibody, indicating that the synthetic peptide had the function of mimetope and could have an immune reaction with anti-rmESP-P1, and TGGFT was a linear BCE.

7 Indirect immunofluorescence detection

After human ejaculated sperm and mouse epididymis tail sperm smears, rabbit anti-peptide antibody and mouse anti-rUU436 antibody were used as primary antibodies for indirect immunofluorescence staining. The results showed that there were specific fluorescent signals in the equatorial segments of human and mouse sperm, which were consistent with the localization of hESP and mESP proteins, respectively. There was no fluorescent signal in other regions of the tail and head, and the result of the serum control before immunization was also negative (Fig. 10, 11, 12). It shows that the antibody produced by the synthetic peptide hESP _98-112 can recognize the natural ESP protein in human and mouse sperm. The anti-rUU436 antibody also had cross-reaction with mouse sperm protein ESP, but had no specific reaction band with human sperm extract.

8 Effect of anti-synthetic peptide hESP _98-112 antibody on mouse sperm-egg interaction

Dilute anti-peptide, anti-rUU36 antiserum and pre-immune serum at different concentrations to treat capacitated mouse sperm, then incubate with zona-removed mouse eggs, and observe the ability of the treated sperm to adhere to eggs and penetrate eggs The change. The average number of bound and fused spermatozoa per egg was significantly reduced after treatment of spermatozoa with 1:10 dilution of antiserum compared with pre-immune sera. Anti-peptide antiserum diluted 1:20 can also inhibit sperm-egg fusion. When the dilution increased, there was no significant effect on the adhesion and fusion of sperm and eggs (Fig. 13, 14, 15, 16). This result indicates that the inhibitory effect of the antibody is concentration-dependent. Compared with the control group, the sperm motility and motility parameters of sperm treated with different concentrations of diluted antiserum and pre-immune serum did not change significantly, and sperm agglutination did not occur.

9 Effects of synthetic peptides hESP _98-112 and UU436 on reproductive function in mice after immunization

33.3% (3/9) of the female mice in the synthetic peptide hESP _98-112 immunized group were infertile, and the infertility rate in the rUU436 immunized group was 44.4% (4/9), while the control group were all fertile. The average litter size of pregnant mice in the immunized group was also significantly lower than that in the control group (Table 1). It shows that the synthetic peptide hESP _98-112 and rUU436 protein produce anti-fertility effect after immunizing animals.

Table 1 Effects of synthetic peptide hESP _98-112 and recombinant protein rUU436 on fertility after immunization of BALB/c female mice *p<0.05

Example 2

Identification of cross-reactive antigens between UU474 protein of UU and human sperm protein IZUMO

1. Experimental animals and materials:

Sexually mature male and female BALB/c mice (provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences) with reproductive history were used for both in vivo and in vitro reproductive experiments in mice.

Male New Zealand white rabbits aged 6 months (about 2.5 kg in weight) were purchased from the Animal Science Department of Shanghai Jiao Tong University School of Medicine.

2. Main reagents:

UU liquid medium and solid medium (Shanghai Tongji Hospital Ministry of Railways Key Laboratory of STD); restriction enzymes BamH I, Hind III, EcoR I, T4 DNA ligase, gel recovery DNA kit, reverse transcription kit, La Taq enzyme (Dalian Bao Biological Company); pre-stained low molecular weight standard protein marker, low molecular weight standard protein marker (Fermentas); Agorose (Promega); plasmid mini-extraction kit (Shanghai Huashun Company); Ni ²⁺ - His column (Qiagen); BCA protein quantification kit (Pierce); ECL fluorescent stain, PVDF membrane, antibody purification PROSEP-A Kit (Millipore); complete and incomplete Freund's adjuvant, anti-hCD46 Monoclonal antibody, Rhodamine (TRITC) goat anti-mouse IgG, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG, FITC-labeled goat anti-rabbit IgG, pregnant horse serum (PMSG), M16 fluid, human chorion Gonadal hormone (hCG), hyaluronidase, proteinase K, Hoechst33342, A23187 (Sigma); acrylamide, bisacrylamide, Tween-20, TEMED, Tris, BSA, sodium chloride, antibiotics and other general chemical reagents were purchased from Shanghai Chemical Reagents Ltd.

3. Main instruments:

ND-1000 UV spectrophotometer (NanoDrop), Mastercycler ep series PCR instrument (Eppendorf), Anthos Zenyth 1100 Multimode microplate reader (Anthos), gel image analysis system, Mini Protean 3 system SDS-PAGE electrophoresis instrument and Mini Semi- Dry TransBlot transfer instrument, 1000/500 electrophoresis instrument and transfer instrument (Bio-Rad), J2-21 high-speed centrifuge (Beckman), LSM-510 laser confocal upright two-photon microscope (Carl Zeiss).

2.2.2 Experimental method

2.2.2.1 UU cultivation:

① Resuscitate the UU specimen frozen at -20°C in a 37°C water bath.

② Take 100μl and inoculate it into 1ml UU liquid medium containing 1% phenol red and 0.1% urea at pH 5.5～6.5, incubate at 37℃ for 16～24h, observe the color change, the indicator changes from orange to orange red, and the culture medium Clarified, indicating UU growth.

③Take 100 μl of UU suspension and inoculate it in solid medium with 95% air and 5% CO ₂ . After culturing in a 37°C incubator for 36-72 hours, the colonies are observed under a low-power microscope to look like "fried eggs".

④ After being positively cultured in liquid medium at 37°C for 16-24 hours, passage in liquid medium for three times to amplify.

2.2.2.2 Mass spectrometric identification of the Ig-like domain (PB) protein of the recombinant Izumo protein:

① The purified recombinant PB protein was subjected to SDS-PAGE electrophoresis.

② Coomassie brilliant blue staining and decolorization.

③ Identification by MALDI-TOF/TOF mass spectrometry (Finnigan LCQ Mass spectrometer 4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA).

2.2.2.3 Use the Western blot method to identify whether the cross-reactive antigen between Izumo and UU is a B cell epitope:

①Design 3 pairs of complementary nucleotide gene sequences according to the peptide sequences of 3 cross-reactive antigens Izumo _212-216 (KGKEA), Izumo _216-220 (ATLTK), and Izumo _220-224 (KPMVG), and design EcoRI/HindIII respectively. Sticky ends, 3 pairs of complementary nucleotide gene sequences are as follows:

Izumo _212-216 (KGKEA) sense strand: aattcg-AAGGGTAAGGAGGCCTAA-ta

Antisense strand: agctta-TTAGGCCTCCTTACCCTT-cg

Izumo _216-220 (ATLTK) sense strand: aattcg-GCCACCCTTACCAAGTAA-ta

Antisense strand: agctta-TTACTTGGTAAGGGTGGC-cg

Izumo _220-224 (KPMVG) sense strand: aattcg-AAGCCAATGGTTGGTTAA-ta

Antisense strand: agctta-TTAACCAACCATTGGCTT-cg

②Double enzyme digestion of pTSA18-Stv plasmid: Reaction system: 10×Buffer M 1.5μl, EcoRI 1μl, HindIII 1μl, pTSA18-Stv plasmid 11.5μl, the total reaction system is 15μl; reaction procedure: incubate at 37°C for 2h; Glycogel electrophoresis verification; target fragments were recovered with a gel recovery kit.

③ Annealing of the three pairs of nucleotide primers synthesized: the sense strand and the antisense strand were diluted with deionized water to 5 μmol/l; annealing reaction: 20 μl sense strand primer, 20 μl antisense strand primer, incubated at 99°C for 5 minutes, and naturally cooled to room temperature.

④ Ligation of the annealed product with the double-digested pTSA18-Stv plasmid: 1 μl of the double-digested pTSA18-Stv plasmid, 2 μl of the annealed product, 1.5 μl of Ligation Solution Buffer, 1 μl of T4 DNA ligase, and 9.5 μl of dd H ₂ O; the total reaction system is 15 μl; reaction program: overnight at 16°C.

⑤ Transform into E.coli DH5a competent bacteria: transform the ligation product into DH5a competent bacteria, spread the transformed product evenly on the LB culture plate containing Kan; culture at 37°C for 12-14 hours; pick positive clones into 5ml LB medium ( Kan) was added; the test tube was incubated overnight at 300 rpm in a shaker at 37°C; the amplified plasmid was extracted with a plasmid mini-extraction kit.

⑥Transformation into E.coli BL21(DE3) competent bacteria: Take 1 μl of small-pumped plasmid and transform into BL21(DE3) competent bacteria, and spread the transformed product evenly on LB dishes containing Kan; incubate at 37°C for 12-14 hours; In the ultra-clean workbench, pick out positive monoclonal bacteria into a test tube that has been added with 5ml LB medium (with Kan added); incubate the test tube in a shaker at 300rpm at 37°C for 5h; amplify the plasmid with a small amount of plasmid Extraction kits were used for extraction; small plasmids were verified by PCR and then sent to Shanghai Huada Gene Company for sequencing.

⑦ Induced expression of Stv-Izumo (Izumo _212-216 , Izumo _216-220 , Izumo _220-224 ) fusion protein: Take 100 μl of each bacterial solution verified by sequencing and add it to a test tube of 5 ml LB medium (with Kan added); Incubate in a shaker at 300rpm at 37°C for 3h, the measured OD value is 0.5, take out 1ml of the bacterial solution in the ultra-clean workbench as a control before induction; add 4μl IPTG to the remaining bacterial solution, and in the shaker at 280rpm, After incubation at 37°C for 4 hours, take 1ml of the induced bacterial solution; centrifuge the pre-induced and post-induced bacterial solutions at 13,000 rpm for 30 sec; discard the supernatant after centrifugation, and add 300 μl dd H ₂ O to resuspend; take the whole bacterial solution before induction and the whole bacterial solution after induction Add 20 μl of 5×sample buffer to 80 μl of each solution, incubate at 95°C for 10 minutes to denature, load the sample, and perform SDS-PAGE electrophoresis (120V voltage) until the dye reaches the bottom of the gel; When the blue band is clear, observe the induction effect according to the protein molecular weight marker.

①Western blot: take Stv-Izumo (Izumo _212-216 , Izumo _216-220 , Izumo _220-224 ) fusion protein as a sample, and perform SDS-PAGE electrophoresis; after electrophoresis, remove the gel, remove the stacking gel, and wet transfer to the membrane Soak in buffer for 15min; soak PVDF membrane in methanol for 15sec, rinse twice with ddH ₂ O, immerse in wet transfer buffer for 10min; use wet transfer method, 80V constant pressure transfer for 2h; V) Skim milk powder blocks the PVDF membrane at room temperature for 2 hours; uses anti-PB IgG as the primary antibody (prepared with 1% skim milk powder at a ratio of 1:2000), and incubates overnight at 4°C. Wash 3 times with TBST, 10 min each time; secondary antibody: horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20000), incubate at room temperature for 1 h; wash 3 times with TBST, 10 min each time; ECL The color agent is used for color development, and the film is developed by an automatic film processor after exposure.

2.2.2.4 Synthetic peptides

The peptide Izumo _212-227 (including ATLTK) was synthesized in Shanghai Sangong, and its antigenicity was enhanced by hemocyanin (keyhole limpethemocyanin, KLH) coupling.

2.2.2.5 Cloning and expressing UU474 protein and preparing its polyclonal antiserum:

① Design and clone UU474 proteins 465-638 (including the cross-reactive antigen ATLTK), and design primers according to the gene sequence of Ureaplasma parvum serovar 3 str. (gi 13357558) in GenBank.

The UU474 protein primer sequence is as follows:

UU474 protein sense strand primer: 5'GC GGATCC AAT CGT TTG GGA ACT GCT 3'

The underlined part is the BamH I restriction site

Negative strand primer: 5'GC AAGCTT GCT TCT GAT GCG TCA TAA AT 3'

The underlined part is the Hind III restriction site

The expected amplified fragment is 524bp;

②PCR: The total DNA released after UU was boiled for 10 minutes was used as a template; the PCR system was as follows: UUDNA 5μl, 10×PCR Buffer 2.5μl, upstream primer 1μl, downstream primer 1μl, dNTPMixtrue (200mM) 0.5μl, dd H ₂ O 14μl, Taq Enzyme 1μl; total reaction system 25μl; PCR reaction program: 94°C pre-denaturation for 5min; 94°C for 1min, 57°C for 1min, 72°C for 1min, cycle 30cycles; 72°C for 10min, 4°C for 10min; PCR product was treated with 1.2% agarose After gel electrophoresis verification, use the gel recovery DNA purification kit to recover.

③Clone: TA clone: Ligation system: pMD-18T Vector 1μl, recovered DNA 4μl, SolutionI 5μl, total reaction system 10μl, reaction procedure: 16°C incubation for 8h; ligation products transformed into DH5a competent cells; screening by X-gal And pick positive clones, and extract the plasmid with a small amount of plasmid extraction kit; plasmid double digestion: 10× Buffer K 1.5 μl, BamH I 1 μl, HindIII 1 μl, small extraction plasmid 11.5 μl (pMD-18T-UU474), total The reaction system is 15 μl; the product recovered from enzyme-digested gel is ligated with pET-28a(+) Vector to construct an expression vector: pET-28a(+) Vector 1 μl, the product recovered from enzyme-digested gel 8 μl, Ligation Solution Buffer 1.5 μl, T4DNA ligation Enzyme 1 μl, dd H ₂ O 3.5 μl, the total reaction system is 15 μl, overnight at 16°C; the ligation product is transformed into E.coli DH5a competent cells; the amplified plasmid is extracted with a plasmid mini-extraction kit; After the plasmid was verified by PCR, it was sent to Shanghai Huada Gene Company for sequencing.

④Expression: Transform pET-28a(+)-UU474 into E.coli BL21(DE3) competent cells; use IPTG, incubate in a shaker at 280rpm, 37°C for 4h to induce the expression of the target protein, and take the whole bacterial solution before induction After induction, add 20 μl of 5×sample buffer to the whole bacterial solution, incubate at 95°C for 10 minutes to denature, load the sample, and perform SDS-PAGE electrophoresis (120V voltage). After clearing, observe the induction effect according to the protein molecular weight marker; according to the above induction expression process, induce 250ml bacterial liquid to express the target protein, and use the Ni ²⁺ -NTA column of Pierce Company to purify the expressed target protein by affinity chromatography; after purification Protein was quantified by BCA method.

⑤Preparation of polyclonal antibodies: Purified recombinant UU474 protein and synthetic peptide Izumo _212-227 were used to immunize a 6-month-old (weight: 2.5kg) male New Zealand white rabbit, and the amount of protein was 0.5-1.0mg per rabbit. To determine the volume of immune protein injection, refer to the literature; use ELISA (enzyme-linked immunosorbent assay) to detect the titer of rabbit antiserum; use PROSEP-A kit for antibody purification.

2.2.2.6 Biological verification of cross-reactive antigens between sperm protein Izumo and UU474 protein:

①Take recombinant PB protein (mouse Izumo protein Ig-like domain) and recombinant UU474 protein as samples, and perform SDS-PAGE electrophoresis; after electrophoresis, remove the gel, remove the stacking gel, and soak in the transfer buffer for 15 minutes; Soak the PVDF membrane in methanol for 15 sec, rinse twice with ddH ₂ O, immerse in the wet transfer buffer for 10 min; transfer the membrane by wet transfer method, transfer the membrane at 80V constant pressure for 2 h; use 5% (W/V) skimmed milk powder in Block the PVDF membrane at room temperature for 1 h; use anti-Izumo _212-227 IgG as the primary antibody (prepared with 1% skimmed milk powder at a ratio of 1:2000), and incubate overnight at 4°C. Wash 3 times with TBST, 10 min each time; secondary antibody: horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:35000), incubate at room temperature for 1 h; wash 3 times with TBST, 10 min each time; ECL The color agent is used for color development, and the film is developed by an automatic film processor after exposure.

② Carry out SDS-PAGE electrophoresis with human sperm protein (the extraction method of human sperm protein is the same as that of mouse sperm protein) as a sample. After electrophoresis, remove the gel, remove the stacking gel, and soak in the transfer buffer for 15 minutes; put the PVDF membrane in Soak in methanol for 15 sec, rinse twice with ddH ₂ O, immerse in the wet transfer buffer for 10 min; transfer the membrane with wet transfer method, 80V constant pressure transfer for 2 h; seal the PVDF membrane with 5% (W/V) skimmed milk powder at room temperature 1h; anti-Izumo _212-227 IgG and anti-UU474IgG were used as primary antibodies respectively (prepared with 1% skimmed milk powder at a ratio of 1:2000), and incubated overnight at 4°C. Wash 3 times with TBST, 10 min each time; secondary antibody: horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:35000), incubate at room temperature for 1 h; wash 3 times with TBST, 10 min each time; ECL The color agent is used for color development, and the film is developed by an automatic film processor after exposure.

2.2.2.7 Human sperm double-labeled immunofluorescence localization:

①Normal human sperm were provided by the Human Sperm Bank of Renji Hospital, and the sperm was collected through the upstream method according to Mandal's method.

②The collected sperm suspension was placed in a 37°C, 5% CO ₂ incubator and incubated for 1.5 hours to achieve capacitation, and then 5 μM Ca ²⁺ inducer A23187 was added to continue incubation for 1 hour to induce sperm acrosome reaction.

③ The spermatozoa after the acrosome reaction were spotted on the glass slides coated with poly-lysine, dried naturally, and washed once with PBS.

④ Block with 5% BSA at room temperature for 1h.

⑤ Anti-Izumo _212-227 IgG (or anti-UU474IgG) and anti-hCD46 were incubated at a dilution of 1:50, and the negative control was incubated with pre-immune serum at 4°C overnight.

⑥ After washing three times with PBS, incubate with FITC-labeled goat anti-rabbit IgG and Rhodamine (TRITC) goat anti-mouse IgG diluted 1:200 in a dark box for 2 hours, wash three times with PBS, and seal with 50% glycerol. slices were observed with a laser confocal microscope.

2.2.2.8 In vitro anti-fertility experiment of anti-UU474IgG and anti-Izumo _212-227 IgG:

①Ovum preparation: 8-10 weeks old female BALB/c mice were intraperitoneally injected with 10 IU of pregnant horse serum (PMSG), 48-72 hours later injected with the same dose of human chorionic gonadotropin (hCG), 12-16 hours later from the oviductal pot Abdominal egg retrieval. The granulosa cell layer around the egg cells was digested with 0.1% hyaluronidase, and the zona pellucida was removed with 0.6% proteinase K.

②Sperm capacitation and antibody treatment: Male BALB/c mice over 10 weeks old were killed by cervical dislocation, and the epididymis tail sperm was taken out and placed in M16 solution containing 0.3% BSA, and the sperm suspension was prepared by the upstream method to adjust the sperm density 1.0×10 ⁶ /ml, incubate at 37°C, 5% CO ₂ incubator for 1.5～2h, add Ca ²⁺ inducer A23187 solution to make the final concentration 5μmol/L, continue to incubate for 1h, use different dilution concentration Anti-UU474IgG or anti-Izumo _212-227 IgG and pre-immune serum were incubated with capacitated sperm for 30min.

③Fertilization: co-incubate zona-free egg cells and differently treated sperm suspensions in a 37°C, 5% _CO2 incubator for 3 hours, suck out the egg cells, remove the sperm loosely attached to the egg cells, transfer to a glass slide, and press Observed under a phase-contrast microscope. After the fusion of sperm and eggs was stained with Hoechst33342, it was observed by laser confocal microscope. The criterion for judging fertilization is: see the male pronucleus or the expanded sperm head and corresponding tail. Fertilization experiments included an experimental group and a control group, and were repeated 3 times.

2.2.2.9 Effect of anti-Izumo _212-227 antibody on mouse sperm motility:

①Incubate with mouse sperm for 1 hour with 50 μg/ml purified anti-Izumo _212-227 IgG or purified normal rabbit serum IgG.

② Sampling was tested by an automatic semen analyzer, and the sperm movement parameters were selected as the percentage of motile sperm (%Motile), curvilinear velocity (curvilinear velocity, VCL), and straight-line velocity (straight-line, VSL).

2.2.2.10 In vivo anti-fertility experiments of UU474 protein and synthetic peptide Izumo _212-227 :

①Select 24 female BALB/c mice and 12 male BALB/c mice, all of which have been verified to be fertile. They were randomly divided into 3 groups, 2 experimental groups (UU474 protein immunization group, synthetic peptide Izumo _212-227 immunization group), and 1 control group.

② Immunize the mice on the first day, the third day, and the 28th day respectively, and each mouse is immunized with 100 μg of protein each time.

③ On the 35th day of immunization, the female and male mice of the experimental group and the control group were caged together, and the cages of both groups were combined with two females and one male.

④ On the 7th day, 14th day and 21st day after closing the cage, the body weights of the female mice in the two groups were weighed. Observe the change of the weight of the female mouse to judge whether the female mouse is pregnant;

⑤ Calculate the litter size of pregnant mice respectively.

2.3 Results

2.3.1 Sequence alignment and analysis of human and mouse Izumo protein Ig-like domains:

In order to further study the physicochemical properties and biological functions of the Izumo Ig-like domain, the sequences of the human and mouse Izumo Ig-like domains were first compared and analyzed by DNAssist 2.0 software. The amino acid sequences of human and mouse Izumo protein Ig-like domains are 64.84% identical and 15.38% similar, indicating that the Izumo protein Ig-like domains are highly conserved among species. Mice can be used as an ideal animal model to study the cross-reactive antigens between human sperm protein Izumo and UU. Through bioinformatics technology, we found that there are cross-reactive antigens between human sperm protein Izumo and UU: KGKEA, ATLTK, and KPMVG are all in the Ig-like domain of human Izumo protein. The results also showed that the mouse Izumo protein Ig-like domain has strong antigenicity and good hydrophilicity.

2.3.2 Mass spectrometry identification of mouse recombinant Izumo protein Ig-like domain (PB):

It was identified by mass spectrometry that the PB protein is indeed the Ig-like domain of the mouse Izumo protein:

2.3.3 Identify the cross-reactive antigen between Izumo and UU by Western blot method:

The pTSA18-Stv-Izumo (Izumo _212-216 , Izumo _216-220 , Izumo _220-224 ) fusion protein was expressed at 11 kDa ( FIG. 17 ). The control protein is Stv-β8 fusion protein, and β8 is an epitope peptide composed of 12 amino acids in β-hCG. In electrophoresis, the molecular weight of the fusion protein is relatively large.

The 212th to 216th amino acid of human Izumo (Izumo _212-216 ) KGKEA, the corresponding amino acid sequence of mouse Izumo is KGKEP, wherein A and P are similar amino acids; the 216th to 220th amino acid of human Izumo (Izumo _216-220 ) ATLTK, corresponding to The amino acid sequence of mouseIzumo is PYLTK, in which T and Y are also similar amino acids; the 220th to 224th amino acid (Izumo _220-224 ) of human Izumo is KPMVG, and the corresponding amino acid sequence of mouseIzumo is KSMVG. Through (Figure 18) we found that anti-PB (Ig-like domain of mouse Izumo protein) IgG can strongly recognize with Stv-Izumo _216-220 fusion protein, but with Stv-Izumo _220-224 and Stv-Izumo ₂₁₂ Weaker recognition occurred for _{the -216} fusion protein. Therefore (Izumo _216-220 ) ATLTK is a relatively strong B cell epitope. The 518th-522th amino acid sequence of the UU474 protein is consistent with the human Izumo _216-220 amino acid sequence, both of which are ATLTK.

2.3.4 Cloning and expressing UU474 protein and preparing its polyclonal antiserum:

The PCR product of the UU474 protein gene was detected by 1.2% agarose gel electrophoresis, and a specific band appeared, which was located between 500 and 600 bp in relative molecular mass (M), consistent with the expected size of 524 bp ( FIG. 19 ).

Under the induction of IPTG (working concentration: 200 μg/ml), the pET-28a(+) expression plasmid was cultured in E.coli BL21(DE3) with T7 RNA polymerase for 4 hours at 37°C, and then ultrasonicated. Separated by SDS-PAGE electrophoresis and stained with Coomassie brilliant blue, the results showed that after induction, the whole bacterial lysate had a band consistent with the expected target protein size at 23kDa (Figure 20), and the expressed protein was in the precipitate after ultrasonic disruption . There was no target protein expression in the whole bacterial lysate before induction.

Use Ni ²⁺ -NTA column to perform affinity layer on the induced bacterial solution. After the eluate was subjected to SDS-PAGE electrophoresis, an obvious band appeared at 23kDa, which was the purified UU 474 protein ( FIG. 20 ).

2.3.5 The titer results of anti-Izumo _212-227 and anti-UU474 rabbit antiserum detected by ELISA method:

The antibody titers obtained were detected by ELISA method. Rabbit serum before and after immunization was used as the primary antibody, and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG was used as the secondary antibody. After color reaction with DAB, the reaction was terminated. , detected at A485 with a microplate reader. The results showed that the IgG titers of anti-Izumo _212-227 and anti-UU474 were both greater than 1:25600 (Table 2), indicating that the obtained antibodies were of high titer (Fig. 21).

Table 2 antiserum titer table

2.3.6 Biological verification of cross-reactive antigens between sperm protein Izumo and UU474 protein:

The cross-reaction between sperm protein Izumo and UU474 protein was verified by Western blot method, and the antibody of synthetic peptide Izumo _212-227 could recognize recombinant PB protein (mouse Izumo protein Ig-like domain) and recombinant UU474 protein (Figure 22) ; anti-UU474IgG or anti-Izumo _212-227 IgG can also recognize human sperm Izumo protein at 37kDa ( FIG. 23 ).

2.3.7 Human sperm double-labeled immunofluorescence localization:

By using purified anti-Izumo _212-227 IgG for double-labeled indirect immunofluorescence localization analysis of human sperm, we found that immunofluorescence localized on the acrosome inner membrane of human sperm after the acrosome reaction, and the positive control antibody CD46 single The localization of the cloned antibodies was consistent ( FIG. 24 ). anti-UU474IgG can also recognize the acrosome inner membrane of human sperm (Figure 25). In the control group, the pre-immune serum was used as the primary antibody, no fluorescence reaction was seen under the confocal laser microscope (Figure 26).

2.3.8 In vitro anti-fertility experiment of anti-UU474IgG and anti-Izumo _212-227 IgG:

The purified anti-UU474IgG, anti-Izumo _212-227 IgG and pre-immune serum diluted with different concentrations were co-incubated with capacitated mouse sperm, and then incubated with mouse eggs without zona pellucida. Changes in the egg's ability to fuse. After Hoechst 33342 staining and observation under a laser confocal microscope, it was found that 50 μg/ml anti-Izumo _212-227 IgG significantly decreased the sperm-egg fusion ability of mice; The mouse sperm-egg fusion ability was not significantly affected ( FIG. 27 ). However, anti-Izumo _212-227 IgG diluted at 25 μg/ml and 10 μg/ml had no significant effect on the mouse sperm-egg fusion ability. The results showed that anti-Izumo _212-227 IgG had a certain inhibitory effect on sperm-egg fusion in vitro.

2.3.9 Effect of antibody on motility function of mouse sperm:

After co-incubating with mouse sperm for 1 hour with purified anti-Izumo _212-227 IgG or purified normal rabbit IgG, samples were taken and detected by an automatic semen analyzer. Compared with the control group, the mouse sperm activity percentage and curve velocity (VCL ) and linear velocity (VSL) were not significantly different, indicating that anti-Izumo _212-227 IgG has no effect on mouse sperm motility (Table 3).

Table 3 Effect of anti-Izumo _212-227 IgG on mouse sperm motility

2.3.10 In vivo anti-fertility experiment of synthetic peptide Izumo _212-227 and recombinant protein UU474:

In order to observe the effects of synthetic peptide Izumo _212-227 and recombinant protein UU474 on reproduction in vivo, female mice were immunized with synthetic peptide Izumo _212-227 and recombinant protein UU474 respectively, and higher titer antibodies could be obtained in the sixth week, and then compared with the already Proven fertile male mice were mated, and the antibodies persisted until the tenth week. Compared with other groups, the litter size of female rats in the synthetic peptide Izumo _212-227 immunized group (Table 4) decreased significantly.

Table 4 Effect of Izumo _212-227 and UU474 immunization on fertilization of female mice

Note: *p<0.05.

Example 3

Study on the function of chimeric peptide immunocontraceptive vaccine

1. Materials Synthesis Peptide CP1 was synthesized by Shanghai Sangon Biotechnology Company.

Complete Freund's adjuvant and incomplete Freund's adjuvant, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG, FITC-labeled goat anti-rabbit IgG, Anti-His monoclonal antibody (Sigma company) PVDF membrane ( Millipore), ECL bioluminescent chromogen (Millipore).

6-month-old (weight about 2.5kg) female New Zealand white rabbits and mature BALB/c mice (8-12W) were purchased from the Experimental Animal Center of the Chinese Academy of Sciences from the Department of Animal Science, Shanghai Jiaotong University School of Medicine.

Second, the method

1. Design and synthesis of chimeric peptides

The cross-reactive antigens _in sperm protein Izumo, tNASP _, and ESP are ATLTK, IERLT, and _TGGFT , respectively. Cross-reactive antigens) are connected in series, and the broad-spectrum T-cell epitope bovine ribonuclease aa94-104: NCAYKTTQANK is connected at its N-terminus, which is used to construct an anti-sperm chimeric peptide vaccine. Chimeric peptide CP1: NCAYKTTQANK GGG TGGFTPEIGK GGG TSIERLTETK GGG KGKEATLTKP. The above 50 peptides were synthesized by Shanghai Sangon Biotechnology Co., Ltd. by solid-phase chemical synthesis.

2. Expression and purification of recombinant mIzumo, mtNASP, mESP-P1 proteins

mESP three fragments P1/P2/P3 clone

Using the extracted BALB/c mouse testicular total RNA as a template, the RT-PCR product was detected by 1.0% agarose gel electrophoresis, and three specific bands appeared, which corresponded to the expected size of the P1/P2/P3 fragments of 516bp, 543bp, 474bp match. The target fragment was connected with the pET-28a(+) vector to construct a recombinant expression vector, which was sequenced again by DNA. Under the induction of IPTG (working concentration: 200 μg/ml), the pET-28a(+) expression plasmid was cultured in E.coli BL21(DE3) at 37°C for 4 hours, and the results showed that the molecular weight of the bacterial liquid after induction was about 25kDa, A large number of new bands are expressed at 36kDa and 21kDa, corresponding to the theoretical molecular weights of P1/P2/P3. The molecular weight will be slightly larger than the theoretical molecular weight, so P1 and P3 are judged to be the target proteins. P2 showed a relatively large molecular weight, and was further verified as the target fragment by mass spectrometry. Under the expression conditions of this experiment, the His-tagged recombinant protein was expressed in the form of inclusion bodies after induction, so the Ni-NTA column was used to perform an affinity layer on the induced bacterial solution analysis. After the eluate was subjected to SDS-PAGE electrophoresis, the results showed that the purified target protein was obtained at the expected protein molecular weight.

Cloning, expression and purification of Izumo protein Ig-like domain and mtNASP

1 Primer:

Izumo gene Ig-like domain (PB) upstream and downstream primers.

Sense strand primer: 5' GC GGATCC AAG CAG TTG CAC ATT TGT C 3' contains restriction site BamH I (underlined part).

Negative-sense strand primer: 5'GC AAGCTT GTT CGG TGG TTG GTT TTC 3' contains restriction enzyme cutting site Hind III (underlined part).

The expected amplified fragment is 336bp;

mtNASP upstream and downstream primers.

The upstream primer 5'GC GGATCC ATGGAACTGCTAGGGCAAGA'3 contains restriction restriction site BamH I (underlined part).

The downstream primer 5'GC AAGCTT TTTGTCTTCAGGTGCTTTCT'3 contains restriction enzyme cutting site Hind III (underlined part).

The target fragment is 339bp in length.

2①PCR: The PCR system is as follows: mouse testis cDNA 5 μl, 10×PCR Buffer 2.5 μl, upstream primer 1 μl, downstream primer 1 μl, dNTP Mixtrue (200 mM) 0.5 μl, dd H ₂ O 14 μl, Taq enzyme 1 μl; the total reaction system is 25 μl.

②Clone: TA clone: Ligation system: pMD-18T Vector 1μl, recovered DNA 4μl, SolutionI 5μl, the total reaction system is 10μl, reaction procedure: incubate at 16°C for 8h; ligation products are transformed into DH5a competent cells; screening by X-gal And pick positive clones, and extract the plasmid with a small amount of plasmid extraction kit; plasmid double digestion: 10×Buffer K 1.5 μl, BamH I11 μl, Hind III1 μl, small extraction plasmid 11.5 μl (pMD-18T-DNA), total The reaction system is 15 μl; the product recovered from enzyme-digested gel is ligated with pET-28a(+) Vector to construct an expression vector: pET-28a(+) Vector 1 μl, the product recovered from enzyme-digested gel 8 μl, Ligation Solution Buffer 1.5 μl, T4DNA ligation Enzyme 1 μl, dd H ₂ O 3.5 μl, the total reaction system is 15 μl, overnight at 16°C; the ligation product is transformed into E.coli DH5a competent cells; the amplified plasmid is extracted with a plasmid mini-extraction kit; After the plasmid was verified by PCR, it was sent to Shanghai Huada Gene Company for sequencing.

③Expression: Transform pET-28a(+)-DNA into E.coli BL21(DE3) competent cells; use IPTG, incubate at 280rpm and 37°C for 4h on a shaker to induce the expression of the target protein, and take the whole bacterial solution before induction After induction, add 20 μl of 5×sample buffer to the whole bacterial solution, incubate at 95°C for 10 minutes to denature, load the sample, and perform SDS-PAGE electrophoresis (120V voltage). After clearing, observe the induction effect according to the protein molecular weight marker; according to the above induction expression process, induce 250ml bacterial liquid to express the target protein, and use the Ni ²⁺ -NTA column of Pierce Company to purify the expressed target protein by affinity chromatography; after purification Protein was quantified by BCA method.

3. Immunization

BALB/c female and male mice with verified fertility were used in this experiment. CP1 was synthesized as an immunogen, and female BALB/c mice were immunized according to the experimental method in the first chapter, and the dose of antigen was 100 μg for each immunization. Mice were immunized in the same way with PBS plus adjuvant as a control. One week after the end of the immunization, the cages were mated for the first time, and the cages were mated again in the 8th week. The cages were separated after 1 week, and the weight of the mice was regularly weighed to determine whether the mice were pregnant, and the number of litters of the pregnant mice was counted. At the same time, a female New Zealand white rabbit was immunized to prepare anti-CP1 antibody for subsequent in vitro penetrating experiments.

4.ELISA

One week after the immunization, blood was collected from the inner canthus vein before the first cage mating, and blood was collected again at the 5th and 8th weeks after the immunization. Coat the 96-well plate with CP1, 1 μg per well, carry out according to the above-mentioned experimental method, and measure the antiserum titer with the pre-immune serum and the serum of the control group as the control. Recombinant proteins rmESP-P1, rmtNASP, and rmIzumo were used to coat 96-well plates, 0.5 μg per well, and 1:100 diluted serum was used as the primary antibody to measure the antibody response of each component epitope in CP1.

5. Human and Mouse Sperm Protein Extraction

Four normal human semen samples were taken to separate and obtain sperm samples, and five adult BALB/c male mice were taken to obtain sperm samples from the tail of the epididymis according to the above method. See Chapter 2 for the experimental method of protein extraction.

6.Western blot analysis

SDS-PAGE was carried out with recombinant mIzumo, mtNASP, mESP-P1 protein and sperm whole protein extract as antigen, rabbit anti-CP1 antibody was used as primary antibody, diluted 1:2000, and Western blot analysis of CP1 induced production was carried out according to the experimental method in Chapter 1 Antibody cross-reactivity with the original protein.

7. In vitro penetration experiment of mouse sperm

The preparation of gametes was the same as before. Dilute CP1 immunized rabbit antiserum to pretreat the sperm at 1:10, 1:20, 1:100, and co-incubate with zona-depleted mouse eggs to observe the adhesion and fusion of sperm and eggs.

(1) Egg retrieval

BALB/c female mice aged 8 to 10 weeks were intraperitoneally injected with 10 IU of pregnant horse serum (PMSG), 48 hours later injected with the same dose of hCG, and 15 to 16 hours later, eggs were collected from the ampulla of oviduct.

(2) Sperm collection and antibody treatment

10-12 weeks old BALB/c male mice were killed by cervical dislocation, the tail of the epididymis was taken out, placed in preheated M16 (containing 3% BSA), the epididymis tissue was slightly cut with surgical scissors, and placed at 37°C, 5% CO _2Stay in the incubator for 10min, prepare the sperm suspension by the upstream method, adjust the sperm density to 1.0×10 ⁶ /ml, incubate at 37°C, 5% CO ₂ incubator for 1h, add Ca ²⁺ to induce A23187 solution, make The final concentration was 5 μmol/L, and the incubation was continued for 20 minutes. Anti-P1, anti-P2, anti-P3 antiserum and pre-immune serum with different dilution concentrations were incubated with capacitated sperm for 30min. Serum was used to inactivate complement at 56°C for 30 minutes before use.

(3) In vitro penetration experiment of mouse sperm

After co-incubating zona pellucida egg cells and differently treated sperm suspensions at 37°C in a 5% CO ₂ incubator for 3-4 hours, aspirate the egg cells, remove the sperm loosely attached to the egg cells, fix with 2% paraformaldehyde for 20 min, After washing with PBS, stain with Hoechst 33342 (10 μg/ml) at 37°C for 15-20 minutes; after washing with PBS, put them on slides and observe with a laser scanning confocal microscope. The number of sperm adhering to the egg membrane was counted under an inverted phase-contrast microscope, and the fusion of sperm and eggs was observed under a fluorescent microscope. The criterion for judging the fusion of sperm and eggs was: the swollen sperm head or pronucleus was seen in the egg cell. The experiment was repeated 3 times.

Anti-synthetic peptide serum and anti-rUU436 serum were inactivated at 56°C for 30 minutes to complement, and incubated with sperm for 30 minutes at different dilutions of 1:10, 1:20, and 1:100. After the treatment, the spermatozoa were co-incubated with the zona pellucida mouse eggs at 37° C., 5% CO ₂ , for 3-4 hours. Sperm loosely attached to the egg cells were removed, fixed with 2% paraformaldehyde for 20 min, washed with PBS and stained with Hoechst 33342 (10 μg/ml) for 15-20 min; washed with PBS, placed on slides, and observed under a laser scanning confocal microscope. The number of sperm adhering to the egg membrane was counted under an inverted phase-contrast microscope, and the fusion of sperm and eggs was observed under a fluorescent microscope. The criterion for judging the fusion of sperm and eggs was: the swollen sperm head or pronucleus was seen in the egg cell. The experiment was repeated 3 times.

8 Statistical Analysis

The in vitro experiments were repeated more than three times, and the results of each group were expressed as mean ± standard error (mean ± SEM). The litter size of mice mating experiments was expressed as mean ± standard deviation (mean ± SD). The difference between the experimental group and the control group was analyzed by group t-test, using SAS6.12 software for statistical analysis, and p<0.05 was considered statistically significant.

Three, the result

1 chemically synthesized peptide

The BCE sequence of CP1 was optimized by DNAstar software, and its hydrophobicity, antigenicity and surface accessibility were analyzed to be strong. CP1 was purified by HPLC with a purity greater than 90%. The charge-to-mass ratio identified by mass spectrometry was consistent with the theoretical value.

Effects of 2CP1 Immunized Mice on Fertility Function

81.8% (9/11) of the female mice were infertile in the first cage mating one week after CP1 immunization, while all of the control group gave birth. In the second cage mating at the 8th to 9th week, the infertile female mice in the experimental group all recovered to give birth, and the average litter size of the pregnant mice was not significantly different from that of the control group (p>0.05) (Table 5).

Table 5 Effects of CP1 immunization on BALB/c female mice on fertility

3CP1 antibody production

The serum absorbance of CP1 immunized mice before and after immunization was measured by ELISA, 96-well plates were coated with CP1, and 1:100 mouse serum was diluted as the primary antibody, which showed that specific antibodies were produced in the immunized mouse serum. There was no immune reaction between pre-immune serum and control serum. There was no significant difference in the level of IgG antibody in the serum of the mice taken at the 1st week, the 5th week, and the 8th week after the immunization ( FIG. 28 ). Interindividual variability in mouse anti-CP1 antibody levels was modest. The serum antibody levels were not parallel to their growth status. No. 1 and No. 9 mice gave birth to 5 and 7 litters respectively, and the rest of the mice were sterile, but the serum antibody levels of No. 1 and No. 9 mice were not significantly lower than other mice . After the rabbit was immunized, the serum anti-CP1 antibody titer reached 1:10000 (Fig. 29).

Antibodies induced by 4CP1 are cross-reactive with recombinant proteins and original proteins in spermatozoa

Recombinant proteins rmESP-P1, rmtNASP, and rmIzumo were respectively coated on 96-well plates, 0.5 μg per well, and serum diluted 1:100 was used as the primary antibody. Results The sera of rabbits and all mice after immunization had immune reactions with rmESP-P1 and rmtNASP proteins, but had no immune reactions with rmIzumo. There was no cross-reaction between the pre-immune serum and the mouse serum of the control group and the above proteins ( FIG. 30 ). Further, the rabbit antiserum was used as the primary antibody to analyze whether the anti-CP1 antibody could recognize the recombinant protein by Western blot. Results Positive bands were seen at the target molecular weights of rmESP-P1 and rmtNASP proteins, which were 25 kDa and 18 kDa respectively, and there was no immunoreactive band at the position of rmIzumo ( FIG. 31 ). The theoretical molecular weights of tNASP in human and mouse spermatozoa are 85kDa and 83kDa, respectively, and the theoretical molecular weights of ESP protein in human and mouse spermatozoa are 38kDa and 45kDa, respectively. The positions of the reaction bands are consistent with the theoretical molecular weights of tNASP and ESP. Preimmune sera did not cross-react with the above proteins (Figure 32). It shows that the BCE of the tNASP and ESP proteins in the CP1 component can induce the production of specific antibodies to recognize the original proteins tNASP and ESP in sperm. There were no immunoreactive bands at the target position of human and mouse natural Izumo protein and rmIzumo, indicating that the antibody could not recognize the original protein or that the BCE of Izumo did not induce the production of specific antibodies.

5CP1-induced antibody inhibits sperm-egg interaction in vitro

Capacitated mouse sperm were treated with different concentrations of diluted rabbit anti-CP1 serum and pre-immune serum, and then incubated with zona-removed mouse eggs, and the changes in the ability of the treated sperm to adhere to eggs and the ability to penetrate eggs were observed. The average number of bound and fused spermatozoa per egg was significantly reduced after treatment of spermatozoa with 1:10 dilution of anti-CP1 serum compared with pre-immune serum. When the dilution increased to 1:20, the number of adhesion and fusion of mouse sperm and eggs was different from that of the control group, but there was no statistical significance (p>0.05). When the dilution increased to 1:100, there was no significant effect on the adhesion and fusion of sperm and eggs (Fig. 33, 34, 35, 36). This result indicated that the inhibitory effect of anti-CP1 antibody was concentration-dependent. Compared with the control group, the sperm motility and motility parameters of sperm treated with different concentrations of anti-CP1 antibody and pre-immune serum did not change significantly, and sperm agglutination did not occur.

In this experiment, the three verified sperm proteins (hIzumo, tNASP, hESP) were combined with the cross-reactive antigen of UU, arranged linearly and connected with a broad-spectrum T cell epitope at the N-terminal to form a chimeric peptide, and immune female BALB/c After immunization, the fertility rate of mice in the immunized group decreased by 81.8%. The three cross-reactive antigens have been verified as linear BCE, which has sufficient antigenicity. The synthetic epitope peptide coupled with KLH alone can induce the body to produce specific antibodies to recognize the original protein, and produce a certain anti-fertility effect. Connecting these three BCEs in series constitutes a new form of vaccine with enhanced immunocontraceptive effects. Antibodies generated by CP1 cross-react with recombinant and native tNASP, ESP proteins. It shows that BCE in the composition of CP1 induces the body to produce specific antibodies, and the important thing is that the antibodies can recognize the original natural protein. It is well known that antibodies induced by peptide vaccines must be able to cross-react with the original natural protein to be functional. There is a view that including several antigenic targets in the chimeric peptide can synergistically enhance the immune effect of the single component BCE. The three sperm antigens based on the peptide vaccine in this experiment are all sperm-specific expressions and are related to fertilization. ESP is located in the equatorial segment of the sperm acrosome and participates in the sperm-egg interaction during fertilization. tNASP is a sperm-specific nuclear autoantigen, and its role in the fertilization process is unclear, but immune challenge of tNASP protein in mice can lead to immune infertility. Izumo is located in the inner membrane of the acrosome and participates in the fusion of sperm and eggs. After gene knockout, it leads to complete infertility, and after immunization, it can also cause partial infertility. It can be speculated that after CP1 immunization in this experiment, the two targets tNASP and ESP involved in the fertilization process of mice were immune attacked, which may be the reason why it can effectively induce mouse sterility.

The BCE-coupled KLH of mIzumo in the components of CP1 can produce specific antibodies when immunized alone, recognizing recombinant and natural mIzumo, indicating that this epitope has strong antigenicity. In this experiment, the anti-CP1 antibody could not cross-react with mIzumo. One possible reason is that the BCE is located at the C-terminus of CP1, which is easily hydrolyzed by endopeptidases or proteases in the body; another possibility is that CP1 forms a new conformation, forming The original protein cannot be recognized because of the secondary structure or the generation of new epitopes. It is also reported in other experiments that when multiple different BCEs are connected in series, some BCEs cannot induce the corresponding antibodies. Hardy et al constructed a multi-epitope vaccine, including immunodominant BCE of PLF, SP56, ZP1 and ZP3, which can cause 50% sterility in mice after immunization, but only induces antibodies that react with SP56 and ZP1. Simply mixing several epitope peptides as a multivalent immunogen, the antigenic peptide components often cannot stimulate the body to produce an immune response. The primary purpose of designing and constructing a multi-epitope chimeric peptide is to use it as an immunogen, which can generate antibodies when embedded in each BCE. The ideal situation for a multivalent vaccine is that each component of the vaccine is sufficiently immunogenic to produce antibodies that recognize the original protein. It is not yet possible to accurately predict the immunogenicity of peptide vaccines, that is, in which conformation the peptide can elicit antibodies that cross-react with the original protein. Combining multiple different epitopes to construct a linear peptide vaccine, trying to separate different epitopes with GGG or GPSL sequences, can improve the flexibility of each epitope to facilitate its induction of immune responses. It is best to optimize the epitope spacing with the software EpiSort to improve the immunogenicity of the polypeptide. Another report branched peptide can improve immunogenicity. The production of each BCE antibody may be related to the position between BCE and TCE, because a pair of chemically synthesized chimeric peptides combined with BCE and TCE studies have shown that BCE is arranged at the C-terminus of TCE to produce anti-BCE antibodies, and vice versa produces anti-TCE antibodies . Of course, if more BCEs and TCEs are combined, the situation must be much more complicated. In addition, whether the embedded epitope can induce antibody production may also be related to the length of the inserted BCE peptide. Since the current chemical synthesis of peptides is limited by the sequence length, designing chimeric peptides to make them suitable for expression in biological systems is also a new topic in the research of biosynthetic multi-epitope peptide vaccines. We are also trying different methods to enhance the immunogenicity of chimeric peptides, such as changing the positions of the three BCEs, increasing the copy number of epitopes, and using prokaryotic expression systems to obtain peptides, so as to further study the production of antibodies to each epitope and the effect of immunocontraception .

As reported by other authors, the sterility induced by the chimeric peptide was reversible. After the last immunization, the sterility was re-mated in 8-9 weeks, and all the female mice induced infertility recovered their fertility. The litter size was similar to that of There was no significant difference between the control group. There was no significant decrease in the IgG antibody titer in the mouse sera at the 1st, 5th and 8th week after the immunization. It is speculated that the recovery of reproductive function in the experimental mice group may be due to the reduction of the local immune response in the reproductive tract, and the serum antibody level has no correlation with the reproductive status of the mice. The antibody level and/or T cell response in the reproductive tract after immunization may be closely related to the reproductive status, but it is difficult to accurately detect the antibody level in the mouse uterus and fallopian tube. Therefore, in order to accurately evaluate the contraceptive effect of an immunocontraceptive vaccine, the first problem that needs to be solved is to accurately evaluate the immune response produced locally in the reproductive tract. Specific IgA and IgG antibodies can now be detected in the oviductal fluid of immunized primates.

Because peptide vaccines generally have weak immunogenicity, better carriers and adjuvants are needed to improve immunogenicity and increase the depth and breadth of immune responses. Broad-spectrum TCEs from different sources have been attempted for the construction of peptide vaccines, thus there is a need for reliable and simple methods for evaluating T cell responses. Freund's adjuvant is commonly used in animal experiments. In this experiment, it can be seen that the local induration and hair loss of the immunized animals are obvious, indicating that the local side effects are large, and it is impossible to be suitable for human use. In addition to designing specific polypeptide immunogens for human experiments, more efficient vaccination methods need to be explored. For example, the polypeptide antigen is inoculated with biodegradable particles, and the adjuvants in the lipid molecule are designed to improve the immunogenicity.

In this experiment, for the first time, chimeric peptides composed of sperm proteins ESP, Izumo, tNASP and UU cross-reactive antigens can produce strong reversible anti-fertility effects after immunizing mice, which is a new way to explore new anti-sperm multivalent immunity. A useful attempt at birth control antigens.

<110>Shanghai Jiaotong University School of Medicine

<120>A synthetic peptide and chimeric peptide for immunocontraception and its application

<160>7

<170>PatentIn version 3.3

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<213> Homo sapiens

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Asn Cys Ala Tyr Lys Thr Thr Gln Ala Asn Lys Gly Gly Gly Thr Gly

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Leu Thr Glu Thr Lys Gly Gly Gly Lys Gly Lys Glu Ala Thr Leu Thr

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<213> Homo sapiens

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Claims (2)

1. the chimeric peptide of an immunological contraception, it is characterized in that: its aminoacid sequence is shown in SEQ ID:1 in the sequence table.

2. the chimeric peptide purposes of immunological contraception according to claim 1 is characterized in that: be used to prepare pregnancy vaccine.

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