A kind of embryo secreted endogenous polypeptide pdbcm and its application
A secretory, endogenous technology, applied in the field of assisted reproduction, which can solve unsatisfactory problems
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Embodiment 1
[0028] 1.1 Screening of secreted endogenous peptides related to blastocyst formation in human blastocyst culture medium
[0029] In order to avoid the mutual interference between embryos, we conduct single embryo culture on human cleavage stage embryos, and collect embryo culture fluid according to the outcome of blastocyst formation (divided into blastocyst formation group and non-blastocyst formation group). The embryo culture medium and the embryo culture medium without the formation of blastocysts were used for peptideomics analysis. A total of 3228 peptides were identified in the results of mass spectrometry analysis, of which 201 peptides were significantly different (T-test, P figure 1 ).
[0030] 1.2 Functional analysis of protein precursors of differential peptides
[0031] In view of the fact that the cell’s endogenous peptides are mainly derived from the degradation of protein precursors by the intracellular proteasome pathway, the IPA software was used to perform functio...
Embodiment 2
[0032] Example 2 The effect of polypeptide PDBCM on mouse preimplantation embryo development
[0033] The HERC2 derived peptide, PDBCM, was synthesized by chemical synthesis. The amino acid sequence is shown in SEQ ID NO. 1. The mouse fertilized egg was cultured with single embryos. Different concentrations (2μg / ml, 20μg / ml) PDBCM polypeptide stimulation: Take 6-8 weeks of ICR mice, inject PMSG, inject hCG after 48h, and take the material after 16h; obtain oocytes from the ampulla of oviduct under a microscope to digest granular cells with hyaluronidase, and manipulate the solution in vitro Wash the naked eggs in M2, place them in 37°C, 5% CO2 balanced KSOM medium, and develop to two cells. In the morning of the next day, the two cells were added to the KSOM culture medium supplemented with different concentrations of PDBCM, and the culture continued until the blastocyst. Observing its effect on the formation rate of mouse blastocysts, it was found that the polypeptide PDBCM ca...
Embodiment 3
[0034] Example 3 Comparison of the content of polypeptide PDBCM in high-quality blastocyst culture medium and non-high-quality blastocyst culture medium
[0035] With the help of PDBCM peptide standards, we compared the content of PDBCM in the culture medium of high-quality blastocysts and non-high-quality blastocysts by mass spectrometry. Figure 4 The results showed that the content of PDBCM in high-quality blastocyst culture fluid was significantly higher than that of non-high-quality blastocysts (the blastocyst score uses the Gamder blastocyst grading method. On the fifth day, blastocysts with a grade of 3BB and above are high-quality blastocysts, and those with a grade below 3BB. Blastocysts are non-quality blastocysts).
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