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A secreted endogenous polypeptide pdff-co3 and its application

A PDFF-CO3, endogenous technology, applied in the field of assisted reproduction, can solve problems such as complex components and unfavorable single factors

Active Publication Date: 2020-10-13
南京医科大学附属妇产医院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the complex composition of the follicular fluid, it not only contains ideal ingredients for promoting oocyte maturation, but also contains substances that inhibit oocyte maturation, such as follistatin, maturation inhibitors, etc., which is not conducive to the study of single factors on oocyte maturation in vitro. Therefore, identifying factors in follicular fluid that promote oocyte maturation and improve oocyte developmental potential to optimize the IVM culture system may bring us more surprises

Method used

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  • A secreted endogenous polypeptide pdff-co3 and its application
  • A secreted endogenous polypeptide pdff-co3 and its application
  • A secreted endogenous polypeptide pdff-co3 and its application

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Experimental program
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Embodiment 1

[0024] In order to exclude individual differences, we collected 5 young IVF patients (patients undergoing IVF treatment due to fallopian tube factors, aged ≤30 years, regular menstruation, normal basic endocrine and AMH) and collected mature follicular fluid (mature group) and antral follicular fluid (mature group) respectively. Follicles that develop asynchronously and need to be punctured in advance during the superovulation process of the antagonist protocol (1.0-1.2cm in diameter, immature group), and use peptidomics to analyze their polypeptide differences. Through mass spectrometry identification, a total of 519 peptides were found in follicular fluid, of which 311 were significantly differentially expressed between the two groups (difference multiple ≥ 2, T-test, P<0.05).

Embodiment 2

[0026] ① NCBI database shows: PDFF-CO3 is derived from CO3 protein chainA,

[0027] (https: / / www.ncbi.nlm.nih.gov / protein / 4I6O_A?report=genbank&log$=protalign&blast_rank=1&RID=NM01ZJ70014). This region also exists on CO3 proteins such as mouse, cow, and pig, so the sequence of PDFF-CO3 is highly conserved in mammals ( figure 1 ). ②ProtParam online analysis found that PDFF-CO3 has an Instability index of 37.1, which is a stable polypeptide and can exist stably; the Aliphatic index is 54.4, and the average Grand average of hydropathicity is -1.256.

[0028] (https: / / web.expasy.org / cgi-bin / protparam / protparam) ③The three-dimensional structure of human and mouse CO3 protein was predicted by pymol software, and it was found that its highly homologous ( figure 2 ); the online database also shows that the CO3 protein domain is highly conserved in various animals ( image 3 ).

Embodiment 3

[0030] The FITC fluorescently labeled peptide of PDFF-CO3 was synthesized by chemical synthesis, added to M16 culture medium (1.5 μM), cultured for 14 hours, and MII stage egg cells were collected, and the nuclei were stained with hoechst (1:500) for 10 minutes. Through laser confocal observation, it was found that the fluorescence was distributed in the perivitelline space and intracellular ( Figure 4 ), indicating that PDFF-CO3 can pass through the embryonic zona pellucida and cell membrane to function.

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Abstract

The invention discloses a secreted endogenous polypeptide PDFF‑CO3 and its application. A secreted endogenous polypeptide PDFF‑CO3, characterized in that the amino acid sequence is shown in SEQ ID NO.1. Application of the secreted endogenous polypeptide PDFF-CO3 of the present invention in in vitro maturation and culture of oocytes. PDFF‑CO3 is a CO3-derived peptide, consisting of 25 amino acids, and there is no related function report yet. Adding PDFF‑CO3 to the egg cell culture medium in the GV stage of mice has the biological function of promoting egg cell maturation; therefore, PDFF‑CO3 can be used in the preparation of drugs for promoting egg cell maturation and improving the developmental potential of egg cells, and in the preparation of culture media.

Description

technical field [0001] The invention belongs to the technical field of assisted reproduction, and relates to a secreted endogenous polypeptide PDFF-CO3 and its application. Background technique [0002] In vitro maturation of oocytes (IVM) is a method of obtaining immature oocytes from the ovary without superovulation or applying a small amount of Gn, and culturing them under conditions adapted in vitro to make the oocytes mature and have the ability to fertilize. Technology. As a hot and cutting-edge technology of assisted reproduction, although it has broad application prospects in many fields such as the treatment of polycystic ovary syndrome (PCOS), ovarian high / low response, ovarian incomplete response, egg donation, and women's fertility preservation However, relevant research statistics show that the current in vitro maturation culture system of oocytes is not perfect, showing a low maturity rate and poor developmental potential. The current clinical pregnancy rate i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N5/075
CPCC07K14/47C12N5/0609C12N2501/998
Inventor 凌秀凤霍然杨烨于晓宁赵纯张军强曹善仁张笑兰
Owner 南京医科大学附属妇产医院
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