Mutated human zona pellucida protein 1, mutant gene, detection methods and applications thereof

A technology for detecting zona pellucida protein and human beings, which is applied in the fields of reproductive engineering, genetics and proteomics, and can solve problems such as zona pellucida defects, decreased fertility, and lack of zona pellucida

Inactive Publication Date: 2015-07-29
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the basis of studies in knockout mice, all zona pellucida proteins (zp1-zp3) in mice appear to play a role in maintaining the zona After that, it will lead to different degrees of zona pellucida defects or even the complete absence of zona pellucida, so that the fertility will be severely reduced or disappeared.

Method used

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  • Mutated human zona pellucida protein 1, mutant gene, detection methods and applications thereof
  • Mutated human zona pellucida protein 1, mutant gene, detection methods and applications thereof
  • Mutated human zona pellucida protein 1, mutant gene, detection methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Detection by agarose gel electrophoresis after PCR amplification of the gene zp1 encoding human egg zona pellucida protein 1

[0044] Due to the unknown cause of primary infertility, the patient underwent artificial insemination with husband's sperm twice in May 2009 and June 2010, but she failed to conceive. In November 2010, I performed IVF-ET in a foreign hospital for assisted pregnancy. The long-term program COH, the initial dose was 225IU / d, and the total number of Gn branches was 27. Everything went well before the egg retrieval. 6 eggs were obtained. , after removing the granulosa cells, it was found that none of the 6 eggs had a zona pellucida, and the egg cells shrank. Three of the patient's siblings were married and all suffered from infertility, and all of them were infertile for unknown reasons.

[0045] Mutation analysis of the zona pellucida gene in a family with infertility caused by the absence of zona pellucida. Take 2ml of blood from the p...

Embodiment 2

[0048] Example 2: Sanger sequencing of the DNA deoxynucleotide sequence of the gene ZP1 encoding human egg zona pellucida protein 1

[0049] Recovery and purification of PCR products: 50-60 μl of PCR products of zp1-4 genes were recovered and purified using Promega's DNA purification kit (Promega: collection tube; Wizard SV Minicolumn; Membrane Bind Solution; Nuclear-free Water). 1% agarose gel electrophoresis to separate the PCR product ~ 50μl. Put on the protective gear, cut off the target band with the gel cutter equipped with the purification kit under ultraviolet light, put it into a 1.5ml EP tube, and weigh the gel. Add 10μl membrane washing solution Membrane Bind Solution (membrane washing solution should be dissolved with 95% alcohol first) for every 10mg of gel, and bathe in 50-65℃ water until the gel is completely dissolved. Transfer the gel solution to the recovery column Wizard SV Minicolumn (the recovery column is placed on the collection tube), and let stand at ...

Embodiment 3

[0052] Example 3: Analysis of the DNA deoxynucleotide sequence PAGE results of the human egg zona pellucida protein 1 coding gene ZP1, showing a deletion mutation

[0053] According to the gene sequence provided by NCBI database (http: / / www.ncbi.nlm.nih.gov / ), Primer Premier5 software was used to design primers for amplifying zp1-7. PCR primers were synthesized by Shanghai Sangon Bioengineering Company (R: CGTGGACTTCAGGCTTCA; F: ATACCTTTGGCAATCCGC). Amplification was completed on Gene Amp970 or PE9600 PCR amplifier. Polymerase chain reaction conditions: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 40s, annealing at 60°C for 40s, and extension at 72°C for 40s, completing 35 cycles; total extension at 72°C for 5 minutes; 4°C. save. 10μL PCR amplification system: d5H2O, 5.8μL; 10×PCR buffer: 1μL; 2.5mmol / L dNTP: 1μL; 25mmol / L MgCl2: 0.8μL; DNA template: 0.8μL (about 150ng); Taq polymerase: 0.4μL ( 2.5U / μL); 20μmol / L primers: 0.1μL each.

[0054] Polyacryla...

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Abstract

The invention belongs to the field of reproduction engineering, genetics and proteomics, and particularly relates to a new mutated human zona pellucida protein 1 (ZP1) and a coding gene thereof. The present invention provides a new amino acid sequence PRT of the mutated ZP1 protein capable of causing human zona pellucida-deleted abnormal phenotype (causing infertility in women), deoxynucleotide sequence DNA of the coding gene ZP1, nucleotide sequence mRNA, and deoxynucleotide sequence cDNA synthesized through reverse transcription of the mRNA, wherein the new amino acid sequence PRT is recorded as mutantZP11-404, the deoxynucleotide sequence DNA is recorded as mutantZP1, the nucleotide sequence mRNA is recorded as mutantZP1 (mRNA), and the deoxynucleotide sequence cDNA is recorded as mutantZP1 (cDNA). The present invention further provides two methods for detecting the mutant gene. According to the present invention, the ZP1 protein and the mRNA can be used for protein expression design in the human zona pellucida-deleted disease mechanism research, and the corresponding animal model can be established; and the DNA can be used for analyzing and identifying whether the pathogenic mutation exists in infertile patients, can be adopted as the target gene of the diagnosis of the infertility female patients, and can provide targeting site for the gene therapy of patients.

Description

technical field [0001] The invention belongs to the fields of reproductive engineering, genetics and proteomics, and specifically relates to a new mutated human egg zona pellucida protein 1 (mutated human ZP1) and its coding gene. In particular, the present invention provides a new amino acid sequence (PRT, 404aa) of the mutant ZP1 protein that can lead to abnormal phenotype of loss of human egg zona pellucida (causing infertility in women), as well as the deoxynucleosides of its coding gene mutated human ZP1 Acid sequence (DNA, 9,324bp) and nucleotide sequence (mRNA, 1,909nt). Background technique [0002] The mammalian zona pellucida refers to a layer of transparent membranous protective layer surrounding the mammalian oocyte, which is composed of 3-4 kinds of glycoproteins (Wassarman, P.M. and E.S. Litscher, Biogenesis of the mouse egg's extracellular coat, the zona pellucida.Curr Top Dev Biol, 2013.102: p.243-66; Avella, M.A., B.Xiong and J.Dean, The molecular basis of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12Q1/68A61K48/00A61P15/08
Inventor 肖红梅
Owner CENT SOUTH UNIV
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