Application of CLRN3 protein as cell surface identification protein for separating X sperms

A cell surface and protein technology, applied in the biological field, can solve the problems of low abundance and few sex-specific proteins, and achieve the effects of small harmful effects, low cost, and small damage

Inactive Publication Date: 2019-08-30
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the hydrophobicity and low abundance of sperm membrane proteins and the limitations of protein identification techniques, few sex-specific proteins have been identified and successfully applied.

Method used

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  • Application of CLRN3 protein as cell surface identification protein for separating X sperms
  • Application of CLRN3 protein as cell surface identification protein for separating X sperms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, the specific expression of CLRN3 protein in X sperm

[0051] 1. Acquisition of sperm total membrane protein

[0052] 1. The sperm sample A and the sperm sample B of the Holstein bull were separated by flow cytometry. Neither sperm sample A nor sperm sample B contained protein of animal origin.

[0053] Sperm sample A contains 120 million X sperm, and the separation purity of X sperm is ≥95%.

[0054] Sperm sample B contains 120 million Y sperm, and the separation purity of Y sperm is ≥90%.

[0055] 2. After completing step 1, use Minute TM Plasma Membrane Protein Isolation and CellFraction Kit extracts the total membrane protein of sperm (sperm sample A or sperm sample B). Specific steps are as follows:

[0056] (1) Take a centrifuge tube (specification: 15 mL), add a sperm sample, centrifuge at 850 g for 20 min at 4°C, and discard the supernatant.

[0057] (2) After completing step (1), take the centrifuge tube, add 5 mL of pre-cooled sucrose aqueous ...

Embodiment 2

[0092] Example 2, predicting the transmembrane structure and subcellular localization of CLRN3 protein

[0093] The antigen suitable for separating X and Y sperm by protein immunization must be a cell surface protein, so that it will not destroy the cell structure and affect the sperm motility after combining with the antibody. Having a transmembrane structure is the basis for ensuring the stability of the antigen. The more transmembrane times, the less likely the cell surface antigen is to escape.

[0094] 1. Predict the transmembrane structure of CLRN3 protein

[0095]The transmembrane structure of CLRN3 protein was predicted by TMHMM Server v.2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ).

[0096] Forecast results see figure 1 . The results showed that the transmembrane times of CLRN3 protein was 4 times.

[0097] 2. Predict the subcellular localization of CLRN3 protein

[0098] WoLF PSORT (https: / / wolfpsort.hgc.jp / ) was used to predict the subcellular localization of CL...

Embodiment 3

[0101] Embodiment 3, Western Blotting detects the expression of CLRN3 protein in bovine X, Y sperm

[0102] TBST buffer: pH 7.5, 20 mol / L Tris-HCl buffer containing 0.05% (v / v) Tween20 and 140 mol / L NaCl.

[0103] 1. Perform 10% SDS-PAGE gel electrophoresis on the total sperm membrane protein (sperm sample A total membrane protein or sperm sample B total membrane protein), and then transfer it to a nitrocellulose membrane. (v / v) Skim milk TBST buffer blocking 1h.

[0104] 2. After completing step 1, add rabbit anti-CLRN3 and incubate overnight at 4°C.

[0105] 3. After completing step 2, wash 4 times with TBST buffer, and then incubate with horseradish peroxidase (HRP)-coupled secondary antibody for 1 h at room temperature.

[0106] 4. After completing step 3, wash 4 times with TBST buffer, and then detect with ECL reagent.

[0107] According to the above steps, replace the rabbit anti-CLRN3 in step 2 with TUBLIN, and keep other steps unchanged, as an internal reference.

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Abstract

The invention discloses an application of CLRN3 protein as cell surface identification protein for separating X sperms. The CLRN3 protein is in specific expression on the surface of the mammal X spermoblast, and an amino acid sequence of the CLRN3 protein is as shown in sequence 2 in a sequence table. The method for separating the X sperms provided by the invention is not only simple and easy to do, and the whole separation process is free from using dye and radiation, the pressure on the spermoblast is small, and the harmful influence on the sperm is relatively small. Through the method provided by the invention, the heavy demand on the sexed sperm in the production can be satisfied. The application disclosed by the invention has important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of CLRN3 protein as a cell surface marker protein in separating X sperm. Background technique [0002] Sex-controlled semen technology for dairy cows is known as the third technological revolution in the rapid multiplication of fine breeds after artificial insemination and embryo transfer. offspring of the intended sex. For dairy cows, insemination is carried out by separating the semen of excellent dairy cows, so as to achieve the purpose of rapid improvement of dairy cow breeds. At the same time, the use of sex-controlled semen significantly increased the proportion of female offspring, which made it possible to increase the intensity of individual selection, thereby accelerating the process of genetic improvement of fine-bred dairy cows. [0003] The separation of X and Y sperm is the key to the technology of sexually controlled semen. In 1980, labora...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/569C12N5/076
CPCC12N5/061C12N2509/00G01N33/56966G01N33/577G01N33/68
Inventor 张胜利沈丹姜力
Owner CHINA AGRI UNIV
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