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Method for analyzing and identifying mammal Y sperm typing by using identification protein

A technology of mammals and sperm, applied in the field of immune analysis, can solve the problems of high cost, sperm damage, high equipment, etc., and achieve the effect of low cost and simple equipment

Inactive Publication Date: 2017-03-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although flow cytometry has improved both in terms of sorting speed and purity, its application in production has been limited by shortcomings or deficiencies such as high equipment, high cost, damage to sperm due to separation, and purchase of intellectual property rights.

Method used

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  • Method for analyzing and identifying mammal Y sperm typing by using identification protein
  • Method for analyzing and identifying mammal Y sperm typing by using identification protein
  • Method for analyzing and identifying mammal Y sperm typing by using identification protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Using immunofluorescence technique to confirm the expression of TSPY protein in bovine sperm acrosome

[0047] Take an appropriate amount of optimized Holstein dairy cow semen and add PBS (phosphate buffer saline, purchased from Hyclone Company) to make the sperm concentration reach 10 5 Individuals / ml, take 100ul semen and smear it on a glass slide (purchased from Wuhan Boster Bioengineering Co., Ltd.) treated with polylysine, and let it air for several minutes (not dry); use a concentration of 0.5% Permeate with TritonX100 / PBS (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) for 5 minutes; then block with 5% bovine serum albumin BSA / PBS for 30 minutes; evenly smear 1% BSA / PBS on the surface of the sample. TSPY protein antibody (purchased from ABclonal Company, Cat. No. A6270) diluted in PBS (1:100 by volume), incubated overnight at 4°C, washed with 1% BSA / PBS for 5min / 3 times; fluorescent secondary antibody goat antibody Rabbit Cy3 dilution ((pu...

Embodiment 2

[0054] 1. TSPY protein is used as Y sperm marker protein, use TSPY protein antibody to separate X sperm and Y sperm, and use flow cytometry to analyze the purity of separated X sperm and Y sperm

[0055] Incubate the optimized Holstein cow sperm with the TSPY antibody in Example 1 (the volume ratio of cow sperm and TSPY antibody is 1:100) at 37°C for 40 min, and then mix it with FITC goat anti-rabbit fluorescent secondary antibody (purchased from Wuhan Google Biotechnology Co., Ltd. company, article number GB22303, volume ratio 1:200) and incubate at 37°C for 40 min, after washing, use a flow cytometer (BD Company of the United States, FACSAria III) to sort out TSPY-positive sperm (that is, Y sperm) and TSPY-negative sperm (that is, for X sperm).

[0056] Utilize the flow cytometer to further test the purity of the X sperm and Y sperm separated above respectively, put the separated X sperm and Y sperm into the flow cytometer (BD Company of the United States, FACSAria III) resp...

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Abstract

The present invention belongs to the technical field of immunoassay, and more particularly relates to a method for analyzing and identifying the mammal Y sperm typing by using an identification protein. According to the present invention, by using immunofluorescence, immunoblotting and flow cytometry analysis, the gene protein TSPY expressed and positioned at the Y sperm acrosome part in the Y sperm is determined, and can be used as the Y sperm identification antigen protein in the separation of the X sperm and the Y sperm of mammal; the Y sperm identification protein TSPY can be used as the Y sperm identification antigen protein in the separation of the X sperm and the Y sperm in the mammal semen, wherein the mammal comprises cow, sheep, pigs, rabbits, horses and the like, and does not comprises humans; and the Y sperm identification protein has advantages of accuracy, rapidness, simpleness, low cost, simple equipment, low harm on sperm and the like in the separation of the X sperm and the Y sperm, and is suitable for animal production.

Description

technical field [0001] The invention belongs to the technical field of immune analysis, and in particular relates to a method for identifying mammalian Y sperm type by using marker protein analysis. Said mammals include cattle, sheep, pigs, rabbits, horses, etc., but not humans. Background technique [0002] In animal husbandry production, the sex ratio of newborn animals has become an important factor affecting the economic benefits of animal husbandry. For example, in dairy farming, it is more desirable to have a higher proportion of dams in the calf. In addition, sex selection can avoid the spread of concomitant diseases in the herd. Therefore, it is very important for animal husbandry to control the sex of the offspring of pregnant dams without affecting the conception rate of dams. However, the efficient and convenient sex control method widely used at present is still being researched and perfected, among which the use of immunological principles and antigen-antibod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/533G01N33/68G01N15/14
CPCG01N33/68C12Q1/686G01N15/14G01N33/533
Inventor 张淑君刘方周浩上官爱哨杨利国南良康梁秋曼
Owner HUAZHONG AGRI UNIV
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