【摘要】：【目的】阿爾茨海默癥治療藥物石杉?jí)A甲(Huperzine A,Hup A)的生物合成途徑起始于賴氨酸脫羧酶(Lysine decarboxylase,LDC)。本研究克隆及表達(dá)了來(lái)源于產(chǎn)Hup A的植物內(nèi)生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR擴(kuò)增法,從一株產(chǎn)Hup A的蛇足石杉內(nèi)生真菌Shiraia sp.Slf14獲得LDC基因,構(gòu)建表達(dá)質(zhì)粒p ET-22b-LDC與p ET-32a-LDC,轉(zhuǎn)化感受態(tài)細(xì)胞E.coli BL21,加入IPTG至終濃度為1×10~( 3) mol/L,于24°C、200 r/min培養(yǎng)8 h,誘導(dǎo)表達(dá)LDC蛋白質(zhì);通過(guò)Ni~(2+)金屬親和層析純化重組LDC并建立酶促反應(yīng)體系,利用TLC檢測(cè)了LDC催化活性。利用生物信息學(xué)軟件分析了LDC的理化性質(zhì)及蛋白質(zhì)的空間結(jié)構(gòu)。【結(jié)果】成功克隆并異源表達(dá)出重組蛋白LDC與Trx-LDC,經(jīng)SDS-PAGE電泳鑒定分子量分別為24.4 k Da和42.7 k Da,與預(yù)計(jì)大小相符。TLC結(jié)果表明LDC與Trx-LDC均具有賴氨酸脫羧酶活性。【結(jié)論】本研究從產(chǎn)Hup A的蛇足石杉內(nèi)生真菌Shiraia sp.Slf14中成功克隆到LDC基因并進(jìn)行了異源表達(dá),檢測(cè)到了其催化活性,為豐富LDC分子信息及闡明內(nèi)生真菌中Hup A生物合成機(jī)制提供參考數(shù)據(jù)。
[Abstract]:[objective] the biosynthesis pathway of Huperzine A Hup A was initiated by lysine decarboxylase (Lysine decarboxylase,LDC) in the treatment of Alzheimer's disease. In this study, the LDC gene of endophytic fungi derived from Hup A was cloned and expressed, and its function was studied. [methods] LDC gene was obtained from an endophytic fungus of Hup A, Shiraia sp.Slf14, by RT-PCR amplification. Expression plasmids p ET-22b-LDC and p ET-32a-LDC, transformant E.coli BL21, were added to IPTG at the final concentration of 1 脳 10 ~ (3) mol/L, at 24 擄C ~ (2) r/min for 8 h to induce the expression of LDC protein. The recombinant LDC was purified by Ni~ (2) metal affinity chromatography and the enzymatic reaction system was established. The catalytic activity of LDC was determined by TLC. The physicochemical properties of LDC and the spatial structure of the protein were analyzed by bioinformatics software. [results] the recombinant protein LDC and Trx-LDC, were successfully cloned and heterogeneously expressed. The molecular weights of the recombinant protein LDC and Trx-LDC, were 24.4 k Da and 42.7 k Da, respectively, identified by SDS-PAGE electrophoresis. TLC results showed that both LDC and Trx-LDC had Lysine decarboxylase activity. [conclusion] the LDC gene was cloned and expressed heterologous from the endophytic fungus Shiraia sp.Slf14, which produced Hup A. The catalytic activity was detected, which provided reference data for enriching LDC molecular information and elucidating the biosynthesis mechanism of Hup A in endophytic fungi.
【作者單位】： 江西師范大學(xué)生命科學(xué)學(xué)院江西省亞熱帶植物資源保護(hù)與利用重點(diǎn)實(shí)驗(yàn)室;江西科技師范大學(xué)生命科學(xué)學(xué)院江西省生物加工過(guò)程重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家自然科學(xué)基金(31460021) 國(guó)家“十二五”重大科技支撐項(xiàng)目(2011BAC13B04) 江西省自然科學(xué)基金(20142BAB214008,20151BAB204003,20151BA204002)~~
【分類號(hào)】：Q78

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