吡咯啉-5-羧酸鹽還原酶3之表達與功能性分析

Abstract

早衰症是一種罕見的遺傳性疾病，基因缺陷造成提早老化的表徵。前人研究顯示，人類吡咯啉-5-羧酸鹽還原酶1(pycr1)基因的突變引起皮膚皺紋等與衰老相關的表徵。PYCR基因有三種，包括Pycr1,2和3。PYCR作用於催化合成脯氨酸的最後步驟，脯氨酸是膠原蛋白中最豐富的氨基酸。因此，研究PYCR基因可能有助了解早衰機制和原因。先前研究集中在Pycr1和Pycr2上，Pycr3的研究很少。我以斑馬魚為實驗模型動物研究pycr3的表達和功能分析。使用RT-PCR及原位雜交，發現pycr3在不同胚胎階段和成魚組織均有表達。此外，使用斑馬魚Pycr3蛋白的N端那一半作為免疫原，製作Pycr3抗體。將含Pycr3重組蛋白的SDS凝膠切片注射到兔子三次，取血清樣品進行滴度活性測試，並在第三次加強注射時犧牲採血。測試抗血清，發現其在表達pycr3的細菌裂解蛋白質中呈現對Pycr3的顯著特異性，但卻無法在斑馬魚胚中偵測到內源性的Pycr3。其可能由於內源性Pycr3過低所導，於是使用硫酸銨沈澱和濃縮離心管來濃縮Pycr3蛋白，但仍無法偵測到斑馬魚的蛋白。目前的結論是該抗體無法辨識斑馬魚胚內源性Pycr3，但此一結論能需進行更多優化試驗始能確定。

Progeria is a rare genetic disease with premature aging phenotypes. Previous studies have found that the mutation of human pycr1 gene can cause aging-related characterization of skin wrinkles. There are three PYCR genes, including Pycr1, 2 and 3. Those PYCRs act on the final step of the catalytic synthesis of proline, which is the most abundant amino acid in collagens. Therefore, studying the PYCR genes may help understand the mechanisms and causes of PYCR-related progeria. Among them, most studies focus on Pycr1 and Pycr2, and little research has been done on Pycr3. In this research, zebrafish was used as a model animal to study the expression and functional analysis of pycr3. By using RT-PCR and whole-mount in situ hybridization, I found pycr3 express in different embryonic stages and adult tissues. Furthermore, I generated a Pycr3 antibody using the N-terminal half of zebrafish Pycr3 GST fusion protein as immunogen. The SDS gel slices containing Pycr3 recombinant protein was injected into a rabbit for three times and serum samples were taken for titer testing and sacrificed at the third boost. I characterized the antisera and found significant specificity to Pycr3 in the pycr3-expressing bacterial lysate proteins. However, the Pycr3 antisera failed to detect the endogenous Pycr3 in zebrafish embryos. It might be due to the lower amount of Pycr3 present in zebrafish embryos. I tried to concentrate the endogenous Pycr3 by ammonium sulfate and concentrated centrifuge tube, but still could not detect the zebrafish protein. The current conclusion is the Pycr3 antibody generated cannot recognize zebrafish endogenous Pycr3, however, more rigorous optimizations are needed to draw a firm conclusion.