五氯酚誘發吳郭魚細胞色素P450之純化及免疫分析

Purification and Immunoassay of Pentachlorophenol-induced Cytochrome P450 in Tilapia (Oreochromis niloticus x O. aureus)

本研究以五氯酚(pentachlorophenol, PCP)來誘發吳郭魚(Oreochromis niloticus×O.aureus)肝臟細胞色素P450 (CYP)，再進一步以色層分析方法進行分離及純化，同時以純化出來的CYP製作多株抗體，並建立免疫分析方法，以作為水產品遭受污染監控的指標測定及魚類對於生體異物代謝研究之用。以注射PCP的方式處理吳郭魚，其肝臟細胞色素P450的含量明顯高於控制組，且其aryl hydrocarbon hydroxylase (AHH)、ethoxycoumarin-O-deethylase (ECOD)及ethoxyresorufin-O-deethylase (EROD)之活性明顯高於控制組，但aniline-4-hydroxylase及aminopyrine-N-hydroxylase的活性則不受影響。將注射PCP之吳郭魚的肝臟微小體(microsomes)以sodium cholate及Triton X-100等介面活性劑抽出後，再依序以Octylamino-Sepharose、diethylaminoethyl (DEAE)-Sepharose及CM-Sepharose、hydroxylapatite等層析法分離，可分離出細胞色素P450的含量為19.11±0.76 nmol/mg，純化倍率達79.17±2.92倍，經電泳分析鑑定為單一的45 kDa蛋白質，以一氧化碳－－差異式光譜分析(CO-difference spectral analysis)可得到其最大吸收波長在448 nm，同時其亦具有AHH、EROD之單加氧酶活性，顯示其為CYP1A-likeprotein。本研究亦嘗試以此CYP1A-like protein免疫小白鼠製備出多株抗體，由實驗結果可知該抗體效價及專一性頗佳，目前正進一步測試此多株抗體之特性與應用性，以利接下來的研究所用。

Pretreatment of tilapia, Oreochromis niloticus×O. aureus, with pentachlorophenol (PCP) increased liver microsomal cytochrome P450 (CYP) contents, and aryl hydrocarbon hydrolase (AHH), ethoxycoumarin-O-deethylase (ECOD), and ethoxyresorufin-O-deethylase (EROD) activity, but had no effects on aniline-4-hydroxylase and aminopyrine-N-hydroxylase. A PCP-inducible cytochrome P450 were purified from liver microsomes. Four steps of column chromatographies (Octylamino-Sepharose, DEAE-Sepharose, CM-Sepharose and hydroxylapatite) were performed consecutively, and the final preparation containing 19.11± 0.76 nmol CYP/mg protein gave a single band of 45 kDa on SDS-PAGE. The protein showed an absorption maximum at 448 nm in CO-difference spectrum and had the catalytic properties of AHH and EROD. These results show that PCP could induce ”CYP1A-like protein” in tilapia liver, which is similar to other teleost CYP1A with respect to spectral and catalytic properties. The purified tilapia CYP1A-like protein was subjected to produce polyclonal antibody. BALB/C mice were immunized intraperitioneally with the protein for four times every four-week. The result shows the titer of anti-CYP1A-like protein was 1/8,800 by ELISA, the antibody reached with the purified tilapia CYP1A-like protein was specifically in immunoblotting.