Application of emc10 protein as a biomarker in the diagnosis of male infertility

A substance, sperm technology, applied in the field of application in the diagnosis of male infertility, which can solve problems such as unclear underlying causes

Active Publication Date: 2020-09-04
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the underlying causes of most of the

Method used

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  • Application of emc10 protein as a biomarker in the diagnosis of male infertility
  • Application of emc10 protein as a biomarker in the diagnosis of male infertility
  • Application of emc10 protein as a biomarker in the diagnosis of male infertility

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1, the expression of EMC10 protein, the preparation of purification and EMC10 polyclonal antibody

[0046] 1. Carrier Construction

[0047] The EMC10 gene was connected with the vector pET32a to obtain the recombinant vector pPROEX HT-EMC10.

[0048] The recombinant vector pPROEX HT-EMC10 was sequenced. According to the sequencing results, the structure of the recombinant vector pPROEX HT-EMC10 is described as follows: the nucleotide sequence of the EMC10 gene is inserted between the EcoRI and HindIII restriction sites of the vector pET32a.

[0049] 2. Expression of EMC10 protein

[0050] (1) The recombinant vector pPROEX HT-EMC10 was introduced into Escherichia coli ROSSET (DE3) to obtain the recombinant Escherichia coli named ROSSET (DE3) / pPROEX HT-EMC10.

[0051] (2) After completing step (1), inoculate a single colony of ROSSET(DE3) / pPROEX HT-EMC10 in 5 mL of YTA medium, culture at 37°C with shaking at 250 rpm until OD 600nm It is 1.0-2.0, and the bac...

Embodiment 2

[0065] Example 2, Emc10 Gene Knockout Causes Male Sterility in Mice

[0066] 1. Acquisition of targeting vectors

[0067] Using bacterial homologous recombination technology, insert two loxp fragments into both sides of exon 2 of Emc10 gene (Ensembl number: ENSMUSG00000008140), and then insert anti-neomycin elements into exon 2 and exon 3 In between, the targeting carrier was obtained. For the construction method of the targeting vector, refer to the following literature: Pentao Liu, Nancy A., Jenkins and Neal G. Copeland. A highly efficient recombineering-based method forgenerating conditional knockout mutations. Genome Res. 2003 Mar; 13(3): 476-84. Chan W , Constantino N, Lee SC, Su Q, Melvin D, Court DL, Liu P. A recombineeringbased approach for high-throughput conditional knockout targeting vector construction. Nucleic Acids Res. 2007; 35(8): e64. Epub 2007 Apr 10.

[0068] 2. Obtaining of Emc10 gene knockout homozygous mice

[0069] (1) The targeting vector obtained in...

Embodiment 3

[0077] Example 3, Emc10 Gene Knockout Causes Phenotypic Study of Male Sterility in Mice

[0078] 1. The Emc10 gene may be involved in the fertilization process of fertilized eggs

[0079] 1. Take 6 male Emc10 - / - Mice were mated with several female wild-type mice for 3 months. During mating, the reproductive tract of female wild-type mice was observed for the presence of sperm plugs.

[0080] The results of the experiment are as follows: there are sperm plugs in the reproductive tract of female wild-type mice, indicating that the mating is normal; but no offspring were born.

[0081] 2. In vitro fertilization experiment 1

[0082] (1) Intraperitoneal injection of 5 units of pregnant mare serum gonadotropin into mature female wild-type mice.

[0083] (2) Forty-eight hours after completing step (1), 10 units of hCG were intraperitoneally injected into the mature female wild-type mice.

[0084] (3) At 13 hours after completing step (2), the mature female wild-type mice were ...

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Abstract

The invention discloses application of EMC10 protein as a biomarker in diagnosis of male infertility. An experiment proves that by adopting substances capable of increasing EMC10 protein activity and / or expression quantity, the male infertility can be prevented, the male infertility can be treated, fertilisation of sperm and eggs is promoted, fertilisation of sperm and zona pellucida-free eggs ispromoted, the mixing ability of the sperm and a cell membrane is improved, sperm dysfunction is treated, sperm maturation is promoted, maturation of the sperm in epididymis is promoted, the motion ability of the sperm is improved, a protein tyrosine phosphorylation level related with capacitation of the sperm is improved, the capacitation of the sperm is promoted, the acrosome reaction ability ofthe sperm is improved, an abnormal form of the sperm is repaired, the activity of sodium / potassium-APT enzyme is recovered, ionic balance in the sperm is maintained, Na<+> balance in the sperm is maintained, HCO3-balance in the sperm is maintained, the balance of a pH value in the sperm is maintained, and the fertility of males is improved. The EMC10 protein disclosed by the invention has an important application value.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of EMC10 protein as a marker in the diagnosis of male infertility. Background technique [0002] Humans first discovered the endoplasmic reticulum membrane protein complex (ER Membrane Protein Complex, EMC) in yeast, which is a complex composed of six subunits (Emc1 protein-Emc6 protein) related to protein folding in the endoplasmic reticulum . Later, in the application of proteomics to study the degradation process of endoplasmic reticulum-related proteins, it was found that EMC is a complex composed of 10 subunits (ie, EMC1 protein-EMC10 protein) in animals. The EMC is a multifunctional, evolutionarily conserved protein complex that is thought to play multiple roles in ER degradation, ER-mitochondria anchoring, and assembly of multi-transmembrane proteins. For example, EMC6 protein may be involved in the degradation of specific proteins in the endoplasmic reticulum ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/00A61P15/08G01N33/68
CPCA61K45/00G01N33/689G01N2800/367
Inventor 王宣春周玉传吴斐熊祖泉陈匡阳王新茹曹心怡李燕良茅善华丁强
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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