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Prostate-specific antigen isomer, coding gene and application thereof

A prostate-specific, gene-encoding technology, applied in the field of new proteases that regulate human fertilization, can solve the problem of male infertility without a clear screening target molecule

Active Publication Date: 2016-08-24
NINGBO BOFENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still no selection standard for whether to use IVF or directly use ICSI in the case of normal semen. In addition, there is no clear screening target molecule in the development of drugs for the treatment of male infertility

Method used

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  • Prostate-specific antigen isomer, coding gene and application thereof
  • Prostate-specific antigen isomer, coding gene and application thereof
  • Prostate-specific antigen isomer, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: The acquisition of novel proteases on human sperm

[0028] 1. Semen collection and sperm collection

[0029] All subjects abstained from sexual intercourse for 3 to 7 days, and the semen was collected by masturbation. The sampling container was all disposable sterile semen cups. After the semen was taken out, it was placed in a constant temperature water bath at 37°C. Computer-assisted semen analyzer (CASA) analysis. The fully liquefied semen was washed according to the standards of the "WHO Laboratory Manual for Human Semen and Sperm-Cervical Mucus Interaction" (Fourth Edition), and the sperm pellet (ie, washed sperm) was collected.

[0030] 2. Preparation of Sperm Extracts

[0031] In the control group (Before wash), 0.5ml of liquefied semen was directly sonicated; in the experimental group (After wash), 0.5ml of Earle's culture medium was added to resuspend the sperm pellet obtained after liquefaction of 1ml of semen in step 1, and then ultrasonicated. U...

Embodiment 2

[0047] Example 2: Isolation and Identification of Novel Proteases on Human Sperm

[0048] 1. Apply RP-HPLC to separate and purify 30kD protease from normal human sperm extract (obtained by the method of Example 1), the chromatographic column selected is: Shim-pack CLC-ODS (4.6mm × 250mm); the solvent contains 0.05 %TFA in acetonitrile; the elution condition is that the solvent concentration is from 0-65%, 60min gradient elution; the flow rate is 1ml / min, and the temperature of the column oven is controlled at 28°C; the eluate is collected in a 1.5ml Eppendorf tube according to the peak time , and then placed in a vacuum concentrator pre-cooled to 4°C and started to concentrate to remove the volatile liquid acetonitrile without concentrating the eluate to dryness; after concentration, it was dialyzed in Earle's culture medium, and the activity of the retentate was detected by gelatin SDS-PAGE, the result See image 3 .

[0049] 2. Through multiple collection, concentration, d...

Embodiment 3

[0051] Example 3: Prediction of protein three-dimensional structure

[0052] The amino acid sequence of PSA (amino acid sequence shown in SEQ ID NO.3) and PSA_h is compared ( Figure 5 In a), like PSA, PSA_h has exactly the same signal peptide of 17 amino acids, activating peptide of 7 amino acids and mature peptide of 186 amino acids at the N-terminus, but the sequence of the C-terminus is very different. There are two cysteines for disulfide bond formation in the C-terminal sequence of PSA, while PSA_h lacks these two cysteines ( Figure 5 In a, △), we speculate that it may form a three-dimensional structure that is quite different from PSA. At present, the three-dimensional structure of PSA has been resolved, and PSA has two distinct β-sheet barrel domains (A1 and A2). The first domain A1 is composed of 7 β-sheets, and the second domain A2 is composed of 6 β-sheets and a long α-helix of 11 amino acid residues. We used the 3D structure online prediction software Protein H...

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Abstract

The invention discloses a prostate-specific antigen isomer, a coding gene and application thereof. The protein has obvious gelatinase activity in vitro; and the deficiency and decrease of the protein are key factors for male infertility, and therefore, the protein can be used as a new index for male infertility. The protein-deficient sperm is capable of completing fertilization, and thus, the protein can also be used as an index for selecting artificially assisted reproduction IVF and ICSI technologies. Besides, the protein is also an important drug screening target molecule for male infertility.

Description

(1) Technical field [0001] The invention relates to a sperm regulating protein, in particular to a novel protease, coding gene and application thereof for regulating human fertilization. (2) Background technology [0002] Fertilization is the fusion of sperm and egg into a zygote, which plays a key role in ensuring the inheritance of both parents. Sperm do not have the ability to fertilize eggs when they leave the testis, and two acquisition processes are required: one is the transformation of proteins and lipids in the sperm plasma membrane and internal structure during the process of crossing the epididymis; Some modification of the surface and interior of spermatozoa during the passage is called capacitation. When the capacitated sperm contacts the egg membrane or the zona pellucida around the egg, it stimulates the acrosome reaction of the sperm and helps the sperm to further pass through the egg membrane. Fertilization occurs in the ampulla of the fallopian tube. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/57G01N33/573
CPCC12N9/6489G01N33/573G01N2333/96488G01N2800/36
Inventor 杨卫军钱叶青
Owner NINGBO BOFENG BIOTECH
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