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Method for detecting expression of 305 sperm localization proteins in fertility-related human testis and epididymis

A technology related to proteins and proteins, applied in the field of detection, can solve problems such as the inability to analyze protein expression levels in seminal plasma, poor understanding of male infertility-related proteins, and limited detection methods

Active Publication Date: 2011-11-30
山东艾胚康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the causes of male infertility are very complex and extensive, the current routine semen detection methods are very limited, making it impossible to systematically analyze the protein expression level in seminal plasma
[0005] To sum up, so far, people’s understanding of male infertility-related proteins caused by human sperm maturation is still not in-depth, and there is a lack of detection methods for such proteins. Therefore, it is urgent to develop effective methods and products for the detection of male fertility-related proteins

Method used

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  • Method for detecting expression of 305 sperm localization proteins in fertility-related human testis and epididymis
  • Method for detecting expression of 305 sperm localization proteins in fertility-related human testis and epididymis
  • Method for detecting expression of 305 sperm localization proteins in fertility-related human testis and epididymis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Epididymis and testis specimens were taken from young males (27-30 years old) who died unexpectedly. They had a history of childbirth and no reproductive system diseases. They had signed an agreement with their family members to donate the body to medical research, and passed the approval of the Ethics Committee of Yantai Yuhuangding Hospital. . Immediately after the specimens were obtained through surgery, the epididymis luminal fluid and testicular tissue protein samples were extracted and quantified, and the samples of more than three individuals were mixed in equal amounts for two-dimensional gel electrophoresis separation, and the common protein spots of three repeated electrophoresis were taken for mass spectrometry identification, among , 305 proteins were newly found to be clearly positioned on sperm.

Embodiment 2

[0080] Example 2 sperm preparation

[0081] Normal semen was liquefied at a constant temperature of 37°C for 30 minutes, and 1.0ml of semen was drawn to equilibrate Earle's liquid at 37°C, 5% CO2 and saturated humidity for 12 hours, and the test tube was inclined at 45° and incubated for 60 minutes to fully mobilize sperm Upstream, centrifuge at 600g for 10min. Discard the supernatant, repeat the washing twice, and set aside. Normal semen was provided by the Reproductive Center of Yantai Yuhuangding Hospital with the consent of the provider.

Embodiment 3

[0082] Example 3 Expression vector construction and protein purification

[0083] The protein products of the above 305 genes were obtained by the following methods. According to the gene sequence, synthesize a pair of specific primers for amplifying the mature coding region. Using the human epididymis cDNA library prepared by conventional methods as a template, the target gene was directly amplified by PCR, and then cloned into a commercially available vector pGM-T vector (purchased from Shanghai Bebo Biotech Co., Ltd.) for sequencing and identification. The gene identified by sequencing was cloned into the expression vector pET32b(+) (purchased from Shanghai Beibo Biological Co., Ltd.) through restriction sites, so that it was consistent with the reading frame of the fusion tag. Transform the recombinant expression vector into conventional E.coli BL21 (DE3) competent cells to obtain engineering bacteria. The expression was induced by 1mM IPTG at 32°C, and the recombinant p...

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Abstract

The invention relates to a method for detecting 305 kinds of semen positioning protein of human testicle and epididymis expression related to human procreation. More specifically, the invention provides an important protein group related to sterility caused by human semen maturation and also provides a qualitative and quantitative method for detecting human semen positioning protein on semen as well as in seminal plasma, thereby providing a new way for definitely diagnosing the causes of male sterility.

Description

technical field [0001] The present invention relates to the field of detection, more specifically to a method for detecting 305 kinds of sperm localization proteins that are related to fertility and expressed in human testis and epididymis, including the qualitative and quantitative methods of using fluorescent, enzyme-labeled, silver-labeled, biotin, isotope, chip and other labeling techniques. A method for quantitatively detecting sperm localization proteins. The present invention also relates to a protein chip for detecting the sperm localization protein and its application Background technique [0002] Male infertility is one of the important factors causing infertility. According to the World Health Organization (WHO) report in 2000, global infertility accounts for about 15%, and male factors account for about half; in some European countries, infertility is as high as 30%. The causes of male infertility are very complex and extensive, involving abnormalities and obst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N2800/34G01N33/6893G01N33/689G01N2800/367
Inventor 李建远
Owner 山东艾胚康生物科技有限公司
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