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Purposes of epididymis characteristic antibiotic peptide Bin1b in spermiotiliosis

A sperm maturation, bin1b technology, applied in the fields of peptide/protein components, medical preparations containing active ingredients, pharmaceutical formulas, etc., can solve the problems of no discovery or disclosure of correlation

Inactive Publication Date: 2005-11-02
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But no correlation between Bin1b and sperm maturation has been found or published

Method used

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  • Purposes of epididymis characteristic antibiotic peptide Bin1b in spermiotiliosis
  • Purposes of epididymis characteristic antibiotic peptide Bin1b in spermiotiliosis
  • Purposes of epididymis characteristic antibiotic peptide Bin1b in spermiotiliosis

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention discloses the use of epididymis specific beta-defensins, which are used in preparing medicine for promoting the mature of sperm. The optimal beta-defensin is Bin1b. The present invention also provides the sperm mature promoting composition, which contains beta-defensin in safe and effective amount, calcium ion source and 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 . The secretions of the epididymis, produced by specific regions of the epididymis—called caput (head), corpus (body), cauda (tail)—are critical to the maturation of spermatozoa; however, little is known about the molecular mechanisms of this maturation process few. Of the approximately 200 k...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17
Inventor 陈小章张永莲周晨曦萧莅勍郑敏曾丽玲黄巧茵何乐思锺耀华
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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