在酵母菌內建立老鼠抑制食慾激素Peptide YY (PYY)的表現系統

The establishment of Saccharomyces cerevisiae expressive system of the rat hormone peptide YY (PYY) able to inhibit appetite

蕭垂鑫(Chui-Hsin Hsiao)

指導教授 ： 蔡榮宗

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00099

摘要

Peptide tyrosine-tyrosine(PYY)為胰多肽蛋白家族的成員之一，分佈起源於迴腸末端、結腸和直腸，是由36個胺基酸所組成的短鏈多肽物質，由文獻中已經證實，中樞或周邊系統輸注腸道荷爾蒙酪酪肽(PYY)可以抑制食物的攝取，減輕體重。當食縻進入腸道末端時，L細胞受到營養物的刺激，先將此訊號透過迷走神經傳入到達食慾的調節中樞，PYY荷爾蒙也會經由血液運送穿過血腦障壁至下視丘弓狀核，與Neuropeptide Y/Agouti-related peptide (NPY/AgRP)神經元的Y2 receptor結合，抑制NPY神經肽的釋放，相對的促進Pro-opiomelanocortin(POMC)神經元釋放出α-MSH，α-MSH隨即作用於M3/M4接受器(melanocortin receptor)達到抑制食慾的效果。
根據文獻已知PYY對於食慾抑制的臨床測試，在囓齒動物和人類中大都是使用注射的方式，而我們希望將此由體內自然形成、幾無副作用的荷爾蒙開發成口服的使用方式。首先我們使用PCR的技術將含有限制酶切位的PYY重組基因製造出來並轉殖入pRS406ΔA的質體中，利用colony PCR挑選含有pRS406ΔA-PYY的可能菌株，並且進行DNA定序，確認其序列的正確性。接著，以限制酶將含有重組基因的PYY切下轉殖到pET28載體中，獲得pET28-PYY，接著在大腸桿菌中大量表達及純化PYY重組胜肽。然後，再將N端含有His蛋白標籤的完整PYY重組基因以PCR方式從pET28-PYY上面將其轉殖到酵母菌(Saccharomyces cerevisiae)表現載體pRS424-Gal中，成功建構出pRS424-Gal-PYY表現質體。
未來的研究工作，將根據本研究結果，進一步將純化的PYY重組胜肽及讓可食用的酵母菌大量產生PYY重組胜肽，分別以注射及口服的方式測試其抑制老鼠食慾的成效，開發以酵母菌作為生物性膠囊的評估。

關鍵字

酪酪肽重組基因 ； 血腦障壁 ； 下視丘弓狀核

並列摘要

The gut hormone Peptide YY (PYY) is a member of PP-fold peptide family. PYY is secreted from entero-endocrine L-cells and so named due to the presence of a tyrosine residue at each terminus of the polypeptide(36 amino acid). When dissolved food enter the end of the intestine tract, L-cell, subjecting to the stimulation of nutrients, would transmit the signal through vagal afferent to the appetite regulating center. Also, L-cell can produce PYY hormone. This hormone will flow with the blood, pass through blood-brain barrier (BBB), conduct to hypothalamic arcuate nucleus, bind to Y2 receptor, and inhibit the secretion of NPY/AgRP neuropeptide. Then, Pro-opiomelanocortins(POMC) neurons would release α-MSH that bind to M3/M4 receptor (melanocortin-3 receptor) and result to appetite inhibition.
According to the documents about PYY focusing on the clinical trials to inhibit appetite, and we found that injection is generally used as the method of clinical trials on rodents and human. However, we hope to develop the method by eating the hormone naturally formed from body and expect it to be little side-effect. We used the PCR technique to prepare a PYY recombinant gene (about 135bps). Next, the PYY recombinant gene was cloned into pRS406ΔA vector to construct the pRS406ΔA-PYY plasmid. After sequencing, We confirm PYY gene. Rstriction enzymes cut pRS406ΔA-PYY plasmid at very specific locations and we prepare the insert. Cloning PYY recombinant gene into pET28 vector of Escherichia coli (the resulting plasmid was called pET28-PYY). The recombinant pET28-PYY was transformed into E. coli (BL21 cell). Then, we perform Ni-column purification of recombinant PYY. The His-PYY were confirmed using Western bolt analysis with anti-His antibodies. Moreover, we would use PCR technique to transfer the recombinant gene, containing complete PYY, from pET28-PYY to yeast expressing vector pRS424-Gal.We have constructed the yeast expressive plasmid , pRS424-Gal-PYY.
In future, the ability of purified recombinant PYY and recombinant PYY expressing yeasts to inhibit rat appetite will be assessed by injection or eating respectively. These results will form the basis for the assessment of developing saccaromycete into biological capsule.

並列關鍵字

peptide tyrosine-tyrosine ； recombinant gene ； blood-brain barrier ； hypothalamic arcuate nucleus

參考文獻

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