哺乳類動物胚胎著床前關鍵蛋白質之抗體製備及蛋白質體研究

The Antibody Preparation and Proteomic Studies of the Mammalian Preimplantation Embryonic Key Proteins

沈志昱(Chih-Yu Shen)

指導教授 ： 莊榮輝

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.00595

摘要

在哺乳類動物早期胚胎發育過程中，有數個重要的轉變時期，其中由母源基因轉換至胚源基因的過程，是影響胚胎能否正常發育至囊胚期的第一個重要關卡。過去的研究多半是利用微陣列去分析轉換前後胚胎的所有基因表現，而鮮少有蛋白質層次的研究成果。本論文自豬胚胎 cDNA 資料庫中，鎖定三種共同存在於小鼠與人類胚胎，且在母源-胚源轉換過程表現量最顯著的蛋白質，嘗試以合成短鏈胜 (月太) 免疫誘發出的抗體，有些能夠辨認合成的胜 (月太) 片段，但均無法辨認胚胎上的目標蛋白質。來自豬胚胎的三個未知身份蛋白質，均已製備出單株化抗體，其中兩個完成腹水大量製備，但以小鼠胚胎測試均無效價，豬胚胎測試則因樣本數不足而尚未進行。另一方面，在二次元電泳膠片上，挖取小鼠二細胞與四細胞期胚胎 19 個蛋白質色點作質譜儀身份鑑定後，對於小鼠胚胎母源-胚源轉換過程的生理反應，已有蛋白質層次的初步整體概念。

關鍵字

母源-胚源轉換 ； 合成短鏈胜 (月太) ； 抗體製備 ； 蛋白質體學

並列摘要

Among the transition stages during mammalian preimplantation development, maternal to zygotic transition (MZT) is the first key check point for embryo to maintain normal cleavage through blastocyst stage. Few researchers addressed the difference of protein expression between these transitional stages; only microarray analysis for the gene expression has been reported if any. In this study, three target proteins expressed remarkably during MZT in mouse and human embryos are used as the antigen (synthetic peptides) for antibody preparation. In addition, three unknown genes from porcine embryos were expressed and also used for antibody preparation. Finally, by using proteomic approaches, the protein patterns between mouse 2-cell and 4-cell embryos were compared. Some of the antibodies induced by synthetic polypeptides were able to binding their target pepitdes. However, none could recognize the original embryonic target protein. On the other hand, we produced monoclonal antibodies against the unknown porcine proteins, and two of them were used to produce ascites. However, these antibodies cannot recognize any protein from mouse embryo. Porcine embryo was not tested. On the two-dimensional elecetrophoresis gel, 19 protein spots reflecting the difference between mouse 2-cell and 4-cell embryo were analyzed by mass spectrometry. The results dipicted the change of the protein profile for the maternal to zygotic transition.

並列關鍵字

Maternal to zygotic transition (MZT) ； Synthetic peptide ； Antibody preparation ； Proteomics

參考文獻

Andersen, K.M., Semple, C.A., and Hartmann-Petersen, R. (2007). Characterisation of the nascent polypeptide-associated complex in fission yeast. Mol Biol Rep 34, 275-281.

Aoki, F., Worrad, D.M., and Schultz, R.M. (1997). Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev Biol 181, 296-307.

Betts, D.H., and King, W.A. (2001). Genetic regulation of embryo death and senescence. Theriogenology 55, 171-191.

Braude, P., Bolton, V., and Moore, S. (1988). Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332, 459-461.

Camous, S., Kopecny, V., and Flechon, J.E. (1986). Autoradiographic detection of the earliest stage of [3H]-uridine incorporation into the cow embryo. Biol Cell 58, 195-200.