心臟I型蛋白質磷酸水解酶重組蛋白單株抗體的製備

Preparation of A Monoclonal Antibody that Recognizes Hepp1

李易達(Yi-Da Li)

指導教授 ： 黃憲斌

國立中正大學/理學院/生命科學系生物醫學研究所/碩士(2016年)

摘要

磷酸蛋白質磷酸水解酶 (Phosphoprotein phosphatase, PPP)是一種蛋白質磷酸水解酶(protein phosphatase)。磷酸蛋白質磷酸水解酶家族中被研究最多的包含I型蛋白質磷酸水解酶 (protein phosphatase 1, PP1)、II-A型蛋白質磷酸水解酶 (protein phosphatase 2A, PP2A)以及II-B型蛋白質磷酸水解酶 (protein phosphatase 2B, PP2B)。在細胞中，PP1的催化次單元會與不同的調節次單元結合，這些調節次單元會把催化次單元標的到亞細胞區室並經由去磷酸化去調控PP1受質的功能。目前已找到超過200種的PP1調控蛋白質。心臟I型蛋白質磷酸水解酶結合蛋白(Heart-specific protein phosphatase 1-binding protein, Hepp1)是我們實驗室利用酵母菌雙雜合(yeast two-hybrid)篩選，在人類心臟細胞中找到的一種PP1調控蛋白，並且發現此蛋白質只存在於人類的心臟細胞中。在這個研究中，我們製備了純化的重組(recombinant) Hepp1，並製作了重組Hepp1的單株抗體(monoclonal antibody)。此Hepp1的單株抗體將來可以運用來辨識只存在於人類心臟細胞內的Hepp1。首先，我們將含有Hepp1的質體pET32a-Trx-Ub-Hepp1轉型(transformation)至E. coli BL21(DE3)。再將此含有質體的E. coli BL21培養於37 ℃的Luria-Bertani(LB) Broth培養基內，並以異丙基-β-D-硫化半乳糖苷(IPTG)誘導其製造大量的Trx-Ub-Hepp1融合蛋白。此Trx-Ub-Hepp1融合蛋白經由鎳螯合瓊脂餹凝膠(Ni2+-Sepharose)管柱層析與強陽離子瓊脂餹凝膠管柱層析純化，再經由酵母菌泛素羧端水解酶(Yeast ubiquitin C-terminal hydrolase -1, YUH-1)作用可切割出Hepp1。最後利用強陽離子瓊脂餹凝膠層析法(SP-Sepharose chromatography)即可純化重組Hepp1。我將純化的重組Hepp1送至科羅耐公司代為製作抗Hepp1單株抗體。所得到的單株抗體，經由西方點墨法(western blot)證實可有效的辨認出重組的Hepp1。

關鍵字: I型蛋白質磷酸水解酶, 心臟I型蛋白質磷酸水解酶結合蛋白, 單株抗體

關鍵字

心臟I型蛋白質磷酸水解酶結合蛋白 ； I型蛋白質磷酸水解酶 ； 單株抗體

並列摘要

Phosphoprotein phosphatase (PPP) is one of the protein phosphatases. The most well studied enzymes in PPP family are PP1, PP2A and PP2B. In cells, the catalytic subunit of PP1 is associated with the different regulatory subunits that target the enzyme to different subcellular compartments where PP1 modulates the functions of its substrates through dephosphorylation. Up to now, more than 200 PP1-regulatory proteins have been identified. Heart-specific protein phosphatase 1-binding protein (Hepp1) is one of PP1-regulatory proteins found within human heart cells in our laboratory by using yeast two-hybrid screen. We identified that Hepp1 only exists in human myocardium. In this study, I prepared the recombinant Hepp1 and the monoclonal antibody that recognizes Hepp1. The Hepp1 monoclonal antibody will be applied to identify Hepp1 in human myocardium in the future. I transformed a plasmid (pET32a-Trx-Ub-Hepp1) into E. coli BL21 (DE3). The transformed E. coli was cultured in a LB Broth medium at 37℃. Then, the Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the expression of Trx-Ub-Hepp1 fusion protein. The Trx-Ub-Hepp1 fusion protein was purified by the Ni2+-Sepharose and SP-Sepharose chromatographies. The Hepp1 unit could be cleaved out from the fusion protein by yeast ubiquitin C-terminal hydrolase-1(YUH-1) and purified by SP-Sepharose chromatography. The purified recombinant Hepp1 was sent to the Kelowna company for preparation of the anti-Hepp1 monoclonal antibody. The Hepp1 monoclonal antibody can effectively recognize the recombinant Hepp1 by western blot.

Keywords: PP1, Hepp1, monoclonal antibody

並列關鍵字

monoclonal antibody ； Hepp1 ； PP1

參考文獻

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