Purposes of epididymis specific antibiotic peptide Bin1b in spermiotiliosis

Abstract

The invention discloses the use of epididymis-specific β-defensins, which are used to prepare drugs for promoting sperm maturation. A preferred beta-defensin is Binlb. Also provided is a composition for promoting sperm maturation, which contains a safe and effective amount of β-defensin, a calcium ion source and a pharmaceutically acceptable carrier.

Description

technical field

[0001] The invention relates to the use of epididymis-specific antimicrobial peptide Bin1b (a beta defensin) in sperm maturation. Background technique

[0002] The epididymis, as a male accessory reproductive organ, has been proven to be involved in the maturation of sperm for many years. However, this maturation process has never been linked to a specific molecule derived from the epididymis 1 .

[0003] Mammalian sperm are produced in the testes, but when they leave the testes they can neither swim nor fertilize eggs. They mature and acquire the capacity for forward motility and fertilization as they pass through the epididymis, which is attached to the testis 1，3 . Epididymal secretions, produced by specific regions of the epididymis—called caput (head), corpus (body), cauda (tail)—are critical to sperm maturation; however, little is known about the molecular mechanisms of this maturation process few. Of the approximately 200 known epididymal proteins...

Examples

Embodiment 1

[0050] Bin1b antibody preparation and immunolocalization on epididymis-derived sperm:

[0051] Using the method described in the examples of WO02 / 68463, the cDNA corresponding to 45 amino acid residues and 135 bp bases of the mature Bin1b gene was obtained, as follows:

[0052] 135bp base cDNA:

[0053] GGAATCAGAAACACCGTGTGCTTCATGCAGCGGGGCCACTGTAGGCTCTTCATGTGCCGTTCTGGGGAGAGAAAGGGGGATATTTGCTCTGACCCCTGGAACAGATGCTGCGTATCCAGTTCCATTAAAAACAGATGA (SEQ ID NO: 1)

[0054] 45 amino acid residues

[0055] GIRNTVCFMQRGHCRLFMCRSGERKGDICSDPWNRCCVSSSIKNR (SEQ ID NO: 2)

[0056] The 145bp fragment was inserted into the PET-28a cloning vector (purchased from Invitrogen). The recombinant protein was expressed and purified with the Novagen pET expression kit (the kit was purchased from Invitrogen). The antibody against the recombinant protein was prepared according to the method described in the reference Hu, Y.X. et al. Get effective polyclonalantisera in one month. Cell Res. 12, 157-160 (2...

Embodiment 2

[0060] Bin1b gene eukaryotic transfection:

[0061] Bin1b gene was cloned in pCMV-tag expression vector. T84 cells at 1×10 5 Cell density spread in 35mm culture dish, when the cell growth is 60-80% confluent, use liposome lipofectin transfection reagent to transfect. After transfection, the cells were screened with the selective antibiotic G418 (concentration 400ug / ml), and the operation process was according to the reference Jiang, J.L. et al. The involvement of HAb18G / CD147 in regulation of store operated calcium entry and metastasis of human hepatoma cells. J. Biol. The method described in Chem.276, 46870-46877 (2001) was carried out.

Embodiment 3

[0063] Promoting effect of Bin1b on sperm maturation

[0064] 1. Experimental method

[0065] Sperm-epithelial cell co-culture

[0066] Immature epididymal head spermatozoa were collected from adult SD rats (3 months old). The head of the proximal epididymis was gently minced and placed in 0.4 ml of incubated sperm culture medium (123 mM sodium chloride, 4 mM potassium chloride, 2 mM calcium chloride, 0.4 mM magnesium sulfate, 0.3 mM disodium hydrogen phosphate, 25 mM carbonic acid Sodium hydrogen, 5mM glucose, 12.5mM sodium lactate, 0.5mM pyruvate, 8ug phenol red, 4mg / ml bovine serum albumin, final pH 7.4, viscosity 310mOsm / kg.) 5 minutes to diffuse the sperm into the medium. The sperm concentration was adjusted to 60 million / ml. Methods for isolating and culturing epididymal epithelial cells were described previously. After confluence of epithelial cells (four days after culture), the culture supernatant was removed two hours before co-culture cell experiments. 3 microl...

technical field

[0001] The invention relates to the use of epididymis-specific antimicrobial peptide Bin1b (a beta defensin) in sperm maturation. Background technique

[0002] The epididymis, as a male accessory reproductive organ, has been proven to be involved in the maturation of sperm for many years. However, this maturation process has never been linked to a specific molecule derived from the epididymis 1 .

[0003] Mammalian sperm are produced in the testes, but when they leave the testes they can neither swim nor fertilize eggs. They mature and acquire the capacity for forward motility and fertilization as they pass through the epididymis, which is attached to the testis 1，3 . Epididymal secretions, produced by specific regions of the epididymis—called caput (head), corpus (body), cauda (tail)—are critical to sperm maturation; however, little is known about the molecular mechanisms of this maturation process few. Of the approximately 200 known epididymal proteins...

Examples

Embodiment 1

[0050] Bin1b antibody preparation and immunolocalization on epididymis-derived sperm:

[0051] Using the method described in the examples of WO02 / 68463, the cDNA corresponding to 45 amino acid residues and 135 bp bases of the mature Bin1b gene was obtained, as follows:

[0052] 135bp base cDNA:

[0053] GGAATCAGAAACACCGTGTGCTTCATGCAGCGGGGCCACTGTAGGCTCTTCATGTGCCGTTCTGGGGAGAGAAAGGGGGATATTTGCTCTGACCCCTGGAACAGATGCTGCGTATCCAGTTCCATTAAAAACAGATGA (SEQ ID NO: 1)

[0054] 45 amino acid residues

[0055] GIRNTVCFMQRGHCRLFMCRSGERKGDICSDPWNRCCVSSSIKNR (SEQ ID NO: 2)

[0056] The 145bp fragment was inserted into the PET-28a cloning vector (purchased from Invitrogen). The recombinant protein was expressed and purified with the Novagen pET expression kit (the kit was purchased from Invitrogen). The antibody against the recombinant protein was prepared according to the method described in the reference Hu, Y.X. et al. Get effective polyclonalantisera in one month. Cell Res. 12, 157-160 (2...

Embodiment 2

[0060] Bin1b gene eukaryotic transfection:

[0061] Bin1b gene was cloned in pCMV-tag expression vector. T84 cells at 1×10 5 Cell density spread in 35mm culture dish, when the cell growth is 60-80% confluent, use liposome lipofectin transfection reagent to transfect. After transfection, the cells were screened with the selective antibiotic G418 (concentration 400ug / ml), and the operation process was according to the reference Jiang, J.L. et al. The involvement of HAb18G / CD147 in regulation of store operated calcium entry and metastasis of human hepatoma cells. J. Biol. The method described in Chem.276, 46870-46877 (2001) was carried out.

Embodiment 3

[0063] Promoting effect of Bin1b on sperm maturation

[0064] 1. Experimental method

[0065] Sperm-epithelial cell co-culture

[0066] Immature epididymal head spermatozoa were collected from adult SD rats (3 months old). The head of the proximal epididymis was gently minced and placed in 0.4 ml of incubated sperm culture medium (123 mM sodium chloride, 4 mM potassium chloride, 2 mM calcium chloride, 0.4 mM magnesium sulfate, 0.3 mM disodium hydrogen phosphate, 25 mM carbonic acid Sodium hydrogen, 5mM glucose, 12.5mM sodium lactate, 0.5mM pyruvate, 8ug phenol red, 4mg / ml bovine serum albumin, final pH 7.4, viscosity 310mOsm / kg.) 5 minutes to diffuse the sperm into the medium. The sperm concentration was adjusted to 60 million / ml. Methods for isolating and culturing epididymal epithelial cells were described previously. After confluence of epithelial cells (four days after culture), the culture supernatant was removed two hours before co-culture cell experiments. 3 microl...

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