Lectin composition for detecting male fertility and kit of lectin composition

A technology of lectin and composition, which is applied in the field of lectin composition and its kit for detecting male fertility, and can solve problems such as no analysis

Inactive Publication Date: 2013-12-25
SHANGHAI INST OF PLANNED PARENTHOOD RES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is no comprehensive analysis of the sugar structure map on the surface of human sperm with the lectin chip method, and the screening of differentially bound lectins on the surface of male sperm

Method used

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  • Lectin composition for detecting male fertility and kit of lectin composition
  • Lectin composition for detecting male fertility and kit of lectin composition
  • Lectin composition for detecting male fertility and kit of lectin composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 , human sperm collection and fluorescent labeling

[0017] Step (1) Grouping of semen samples

[0018] Genomic DNA was extracted from male whole blood or sperm, and the rs140685149 site of the DEFB126 gene coding region was genotyped according to the following primer sequences and fluorescent quantitative PCR conditions, and the semen samples were divided into wild-type group and heterozygous group and pure mutation group, amplified with 2W / W and 2D / D primers respectively in two qPCR tubes, and analyzed the data after amplification; the specific formula of qPCR reaction system is shown in Table 1, and the qPCR amplification procedure is shown in Table 2 shows:

[0019] The forward primer of 2W / W_169bp: the nucleotide sequence shown in SEQ ID NO:1;

[0020] The reverse primer of 2W / W_169bp: the nucleotide sequence shown in SEQ ID NO:2;

[0021] The forward primer of 2D / D_295bp: the nucleotide sequence shown in SEQ ID NO:3;

[0022] The reverse primer of 2...

Embodiment 2

[0034] Example 2 、 Detection of sperm samples with different genotypes of DEFB126 using lectin chip

[0035] Step (1) Chip reheating: Take the chip out of the 4°C refrigerator and place it at room temperature for 20-30 minutes to evaporate water vapor and restore the temperature;

[0036] Step (2) chip sealing: configure 50mL TBST, slowly add to the tank, place on a shaker at room temperature and shake gently for 1 hour;

[0037] Step (3) Chip washing: wash with 50mL PBST for 10 minutes, then wash twice with PBS for 10 minutes each time, and dry for later use;

[0038] Step (4) Preparation of the chip incubation tank: put the glass slide with the marked array position as a template on the bottom of the chip, then stick the chip incubation tank on the chip according to the position of the array, and press firmly;

[0039] Step (5) Sperm and chip binding: add 200 μL of fluorescently labeled sperm samples to each incubation tank of the lectin chip, and incubate at room tempera...

Embodiment 3

[0043] Embodiment 3, fluorescence microscopy and flow cytometry detection

[0044] Resuspend 5 × 10 in 100 μl PBS 6Sperm cells were resuspended and mixed with 100 μg / ml FITC-MPL and FITC-ABA respectively, placed at 37°C, and incubated in the dark for 30 minutes. Then wash once with 0.5ml PBS, take 20μl smear, observe the fluorescence signal with fluorescence microscope, then analyze the fluorescence intensity with Facs Calibur flow cytometer, and analyze and identify the results with WinMID2.9 software.

[0045] Analysis and evaluation of implementation results

[0046] Through statistical analysis and comparison of the fluorescent signals of the lectin chips of the DEFB126 gene wild-type, heterozygous and homozygous mutant sperm samples, it can be seen that: (1) Among the 91 lectins on the chip, the DEFB126 wild-type sperm samples can be compared with 54 types (such as figure 1 (shown) lectin binding; (2) comparing the lectin binding signals of the three genotype samples...

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Abstract

The invention discloses a lectin composition for detecting the male fertility. The lectin composition comprises two or more of the following lectins including Jacalin, GHA (Gossypium Hirsutum Agglutinin), MPL (Maclura Pomifera Lectin), ACL (Amaranthus Caudatus Lectin), VVL (Vicia Villosa Lectin) and ABA (Agaricus Bisporus). The invention also provides a kit for detecting the male fertility. The kit comprises the lectin composition and a PBS (Phosphate Buffer Solution) cleaning solution. According to the lectin composition, the fluorescence strength is detected through methods of a flow cytometry, a fluorescence microscope, a liquid chip and the like by utilizing the combination of the 6 lectins and the difference of binding strength of sperm samples of different persons so as to evaluate the quality of sperms, the male fertility is evaluated, and the theoretical basis is provided for research of a male infertile molecule mechanism and etiological diagnosis of male infertility.

Description

technical field [0001] The present invention relates to a lectin composition and a kit thereof, in particular to a lectin composition for detecting male fertility and a kit thereof. Background technique [0002] In mammals, when spermatozoa are produced from the testes and pass through the epididymis, their surface is covered with high-density sugar molecules, thus forming a glycocalyx with a thickness of 20-60nm. Most of the sugar residues are attached to proteins. These sugars Some proteins are integrated on the sperm membrane, and some are loosely connected to the surface of the sperm. At present, the structural map of the glycocalyx on the surface of human sperm is unknown, and only the structure of the sperm membrane glycocalyx is known to be closely related to male fertility. In cynomolgus monkeys, highly glycosylated β-defensin126 (DEFB126), which is secreted from the body of the epididymis and covers the entire sperm surface, is the main glycoprotein component of th...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 施惠娟陶生策辛爱洁周树敏程莉刁华施长根袁瑶吴斌顾一骅于合国王鹏
Owner SHANGHAI INST OF PLANNED PARENTHOOD RES
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