CN102753571B - 参与HEP G2细胞中胆固醇内稳态的大豆7S球蛋白α’亚基延伸区的克隆、酵母表达、纯化和生物学活性 - Google Patents
参与HEP G2细胞中胆固醇内稳态的大豆7S球蛋白α’亚基延伸区的克隆、酵母表达、纯化和生物学活性 Download PDFInfo
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Abstract
将大豆7S球蛋白截短形式的α’链(eα’)克隆并在巴斯德毕赤酵母中表达，所述截短形式的α’链在体内和体外模型中在调控胆固醇和甘油三酯内稳态中有活性。重组多肽跨越从N-端侧的142个氨基酸残基并且包括大豆α’亚基的N-端延伸区。通过常规的生物化学技术纯化eα’多肽并且在人肝细胞瘤细胞系(Hep？G2)中通过监测被标记的LDL的摄取和降解评价所述eα’多肽调节LDL受体活性的潜力。
Description
本发明涉及对应天然α’亚基的N端亲水片段的大豆7S球蛋白的重组α’片段，它的制备方法和含有它的组合物，在所述组合物中它作为控制胆固醇和甘油三酯内稳态有用的活性成分。
发明背景
在高胆固醇血症患者的血脂水平的控制中，膳食大豆蛋白作用的是广为接受的问题[1]。在以前的研究中[2-4]，在体内和体外系统中说明了大豆7S球蛋白的一个亚基，α’亚基，直接涉及LDL-受体的增量调节，表明通过细胞酶加工很可能产生有生物学活性的，能够调节胆固醇内稳态的(多)肽。
天然的7S球蛋白由三个随机组合的、通过不同基因编码的多肽链，α’、α和β亚基组成[5]。成熟的α’(登录号P11827UniProtKB/Swiss-Prot数据库)和α链(登录号P13916UniProtKB/Swiss-Prot数据库)共享约145个氨基酸残基的延伸的N端区，在β亚基(登录号P25974UniProtKB/Swiss-Prot数据库)中缺少所述N端区。基于α’延伸区的特有的氨基酸序列，通过金属亲和色谱纯化此亚基并且将其对高胆固醇血症的大鼠口服施用，因而容许显示它的血浆降脂性质和肝β-VLDL受体的增量调节[4]。在另一方面，由于α’亚基的分子量约为71kDa[6]，它似乎不可能体内穿越肠屏障而没有修饰。
因此，我们的研究指向寻找导致药理学作用的α’亚基的氨基酸序列。由于三个亚基的核心区具有较多的相似的氨基酸序列，可以想到生物学活性应该存在于延伸区的一个或多个(多)肽中。原则上，α’和α链的延伸区之间局部的但显著的氨基酸差异将限制导致生物学活性的肽的数目。
从这些以前的陈述中，寻求的第一策略是检测HepG2细胞中的多肽和合成多肽对LDL-受体(LDL-R)调节的作用，所述HepG2细胞中的多肽获取自CroksoyR70的体外消化(胃蛋白酶/胰蛋白酶)，所述CroksoyR70是在食品治疗高胆固醇血症患者中常规使用的无异黄酮大豆浓缩物[7-8]，所述合成的肽对应于7S大豆球蛋白亚基之间不同的特定氨基酸序列。在这些研究中获得的结果指出，在暴露于CroksoyR70的酶消化产物和来自7S大豆球蛋白的小合成肽(2，271Da)的HepG2细胞中，能够诱导显著的LDL-R增量调节，所述酶消化产物具有在3,000到20,000Da范围内的MW，并以10-4M浓度将所述小合成肽加入细胞[9]。用小肽获得的研究现在仍处于研究中且迄今尚未明确[10]。
大豆蛋白的降低胆固醇和甘油三酯的能力是已证实的问题。大豆蛋白膳食是当今治疗高胆固醇血症患者的最有力的膳食工具，因而为管理成人和非常年轻的对象提供了独特的机会。而且，明确地确立在具有高基线程度的胆固醇血症患者中血浆胆固醇降低更多[14]。
蛋白质本身降低血胆固醇的假说起于实验研究，所述研究表明膳食中从动物到植物蛋白质的转换激活实验动物的肝脏[15]，以及高胆固醇血症患者的循环淋巴单核细胞[16]中的LDL受体系统。为了鉴定导致降胆固醇作用的大豆蛋白组分，用人肝细胞瘤细胞系实施体外研究，所述细胞系对调节LDL受体表达和胆固醇生物合成/分解的因子高度敏感。在HepG2细胞中发现从7S大豆球蛋白纯化的α’亚基增量调节LDL受体[3]并且在胆固醇饲养的大鼠中证实此发现[4]。尽管这些数据支持蛋白质部分导致观察到的生物效应的假说，可对α’链体内生物学命运提出争论，因为通常通过胃和/或肠蛋白分解酶的作用生产肽和氨基酸。然而，声称越来越多的动物和植物(多)肽起相关的调节功能，通常归因于抗氧化、抗增殖和抗炎效应[17]。对大豆而言，实验证据明确地表明了可吸收肽和更小的紧密蛋白质(例如Bowman-Birk抑制物)的可能性[18]，因而诱发很多效应，包括抗癌、抗炎、放射保护性效应[19]。也已经显示了遗传修饰的大豆(多)肽引发生物学应答，比如低血压效应[20]。最近，已经从大豆水解产物鉴定了衍生自7S球蛋白β链的LDL-R转录模拟肽(FVVNATSN)，所述水解产物通过来自解淀粉芽孢杆菌(Bacillusamyloliquefaciens)的蛋白酶并随后通过化学合成制备[21]。在这种情况下，在暴露于100μM浓度肽的HepG2细胞中检测到增加的LDL-R转录(+148％)。已经显示了源自11S球蛋白的其他肽产生相似但是较低的活性[21]。
将需要使得可获得的较短的多肽维持或甚至提高全长蛋白质的生物学性质。
发明描述
现在已经发现所谓的对应其N端侧的大豆7S球蛋白的α’延伸区具有有利的生物学活性并且已被证明在LDL吸收和降解上比全尺寸α’链甚至更有效。
因此本发明提供了所述α’延伸区(在下文中称为eα’)，以及通过克隆、酵母表达和纯化重组多肽制备它的方法，所述重组多肽含有大豆α’亚基的N端延伸区。
为此目的，采用α’链的N端片段的异源表达。在巴斯德毕赤酵母(Pichiapastoris)的分泌-感受态的酵母细胞中达成目标。纯化重组多肽并在HepG2细胞中评价它的生物学活性。通过使用此生物技术方法，能够获得足够量的重组多肽以在体外试验而且也在体内实验中测试。
附图描述
图1pPICZαB-eα’构建体的概观。eα’的表达由AOX(醇氧化酶)甲醇可诱导的启动子(5’AOX1)驱动；α交配因子(α-MF)促进重组蛋白分泌到培养基中；AOX1TT：AOX转录终止区。Shble基因赋予对zeocin的抗性；pUCOri：大肠杆菌中高数量质粒拷贝的复制起点。其他缩写指限制酶的切割位置；bp：碱基对。
图2重组多肽(eα’)和野生型大豆α’亚基的序列比对。星表明两个序列中相同的氨基酸残基。
图2A重组多肽(eα)’的序列(SEQID1)
图2B野生型大豆α’亚基的序列(SEQID2)
图3A重组巴斯德毕赤酵母培养物的还原条件下的SDS-PAGE：
第M道：分子重量标志物
第1道：在用甲醇诱导以前TCA沉淀的培养物上清液(无细胞)。
第2道：在用甲醇诱导1小时以后TCA沉淀的培养物上清液(无细胞)。
第3道：在用甲醇诱导8小时以后TCA沉淀的培养物上清液(无细胞)。
第4道：在用甲醇诱导19小时以后TCA沉淀的培养物上清液(无细胞)。
第5道：在用甲醇诱导25小时以后TCA沉淀的培养物上清液(无细胞)。
图3Beα’纯化步骤的还原条件下的SDS-PAGE
第M道：分子重量标志物
第1道：发酵肉汤的冻干粉
第2道：DEAE-纤维素150mMNaCl洗脱的级分
第3道：DEAE-纤维素250mMNaCl洗脱的级分
发明详述
现在在以下实验部分中将详细描述本发明
材料和方法
酵母、细菌株和化学品。巴斯德毕赤酵母X33(WT)菌株(Invitrogen，SanDiego，CA)用于酵母表达。遗传操作使用的细菌株为大肠杆菌XL1-Blue(Invitrogen，SanDiego，CA)。限制酶PstI和XbaI购自Roche(Indianapolis，IN)，SacI来自Fermentas(Ontario，Canada)TaqDNA聚合酶购自Invitrogen(SanDiego，CA)。Zeocin购自Invivogen(SanDiego，CA)。PCR的寡核苷酸获取自Primm(Milano，意大利)。蛋白胨、胰蛋白胨、酵母提取物和琼脂购自BectonDickinsonandCompany(Sparks，MD)。葡萄糖和山梨糖醇购自Sigma(St.LouisMO)。透析膜购自SpectrumLaboratories(RanchoDominguez，CA)。DEAE树脂购自Whatman(Maidstone，England)。NiNTA树脂购自Qiagen(Hilden，德国)。对称C4HPLC柱购自Waters(Milford，MA)。
其他化学品为来自Sigma(St.Luis，MO)或来自Merck(Darmstadt，德国)的试剂纯。
培养基和生长环境。在YPD(酵母蛋白胨葡萄糖)完全培养基(2％蛋白胨、1％酵母提取物、2％葡萄糖)中培养巴斯德毕赤酵母X33菌株。在含有YPD、1.5％琼脂、100mg/mLzeocin的平板中选择Mut+转化体。将全部酵母培养物维持在30℃。对于异源蛋白质检测，在YPS(酵母蛋白胨山梨糖醇)培养基(2％蛋白胨、1％酵母提取物、2％山梨糖醇)中培养Zeor-Mut+转化体至600nm光密度为5。然后加入甲醇至最终浓度1％。在含有zeocin的低盐LB(Luria-Bertani)培养基(1％胰蛋白胨、0.5％酵母提取物、0.5％氯化钠、1.5％琼脂、25mg/mLzeocin)的平板中选择全部细菌转化体。将全部细菌培养物维持在37℃。
构建eα’基因表达载体。通过PCR在表达巴斯德毕赤酵母质粒DNA模板(pPICZαB)上扩增eα’基因，所述模板含有包含目标序列(eα’)的插入片段。设计寡核苷酸
5’-GAAAAGATAGATTAAAGCTGCAGTGGAGGAAG-3’以在eα’基因的5’端生成PstI限制位点。此突变导致丙氨酸残基插入于eα’的N端位置中。设计又一个寡核苷酸
5’-CCCTTCTTATTCTTTCTAGATCATGGTTCTCTTTGAGACTC-3’以在eα’基因的3’端生成XbaI限制位点。将两个寡核苷酸都溶解于mQ无菌水中。PCR反应混合物由0.5mM引物、0.8mMdNTP(Eppendorf，Hamburg，德国)、30ng模板(pPICZαB/tα’)、2.5UTaqDNA聚合酶、PCR缓冲液(最终组成：50mMKCl、1.5mMMgCl2、20mMTris-Cl、pH8.4和mQ无菌水至最终体积25mL)组成。使用如下条件在PerkinElmerGeneampPCRSystem2400热循环仪(PerkinElmerCorp.，Wellesley，MA)上实施PCR扩增：起始于94℃达10分钟；30个循环的于94℃达40秒，60℃达40秒，72℃达20秒；最终延伸72℃达10分钟并且维持在4℃。酶消化465bp的PCR产物至423bp的全尺寸构建体，并且将它克隆入pPICZαB载体获取pPICZαB-eα’构建体并且转化入XL1-Blue大肠杆菌细胞中。在含有四环素和zeocin的半固态LB培养基上选择阳性克隆。将这些克隆中的一个通过Primm(Milano，意大利)测序以保证在pPICZαB-eα’构建体序列中没有突变发生。通过用限制酶SacI消化将20μgpPICZaB-eα’构建体和30μg表达载体pPICZαB(阴性对照)线性化并然后纯化。
在巴斯德毕赤酵母基因组中转化pPICZaB-eα’。用20μg线性化的pPICZαB-eα’构建体和30μg线性化的pPICZαB在设为1.5kV的Eppendorf(Hamburg，德国)电穿孔仪2510设备上通过电穿孔转化野生型(wt)酵母细胞。首先通过铺平板在含有100mg/mLzeocin的YPD平板上选择转化体。为了证实我们的构建体整合入转化的巴斯德毕赤酵母基因组，使用DneasyPlantMiniKit(QIAGEN，Hilden，德国)以提取基因组DNA。通过使用5’AOX1(5′-GACTGGTTCCAATTGACAAGC-3′)和3’AOX1引物(5′-GCAAATGGCATTCTGACATCC-3′)的PCR，使用基因组DNA作为模板以验证构建体在醇氧化酶启动子(AOX1)位点的插入。PCR反应混合物由0.5mM引物、0.25mMdNTP、100ng基因组模板、2.5UTaqDNA聚合酶、PCR缓冲液(最终组成如上)组成。使用如下条件实施PCR反应：起始于94℃达10分钟；30个循环的于94℃达40秒，60℃达40秒，72℃达20秒；最终延伸72℃达10分钟并且维持在4℃。
转化体克隆选择。在震荡(180rpm)培养器中的30℃50mLYPS培养基中生长18个Zeo+转化体和1个含有pPICZαB表达载体(阴性对照)的转化体至OD600＝5。诱导期通过加入甲醇至最终浓度1％引发并且延长至24小时。通过SDS-PAGE检测上清液等份试样的eα’表达。选择具有最高eα’表达的克隆转化体用于重组蛋白质发酵罐规模的生产。
eα’表达：发酵罐规模。为了大量生产，根据Invitrogen的“毕赤酵母发酵过程指南”(版本B，053002)在14L发酵罐(Chemap，瑞士)中生长选择的克隆。在不带挡板的摇瓶中的YNB培养基(KH2PO42.0g；(NH4)2SO410.0g；MgSO47H2O1.0g；NaCl0.2g；CaCl20.2g；100％甘油10.0g；KOH至pH5.2；蒸馏水至1.0L)上制备接种物培养物。在30℃，250rpm孵育约24小时后，使用1.0L种子培养物接种发酵罐，用8.5L基础盐培养基(BasalSaltMedium)(H3PO485％227mL；CaSO42H2O7.9g；K2SO4155g；MgSO47H2O127g；KOH35g；100％甘油340g；蒸馏水至8.5L；在灭菌后用NH4OH将pH调节至5.0)制备所述发酵罐。用生物素和肌醇(各1mg/L)补充引用的两个培养基。也用修改的PTM1痕量盐溶液(TraceSaltSolution)(H2SO496％2.5mL；CoC2O4°2H2O243mg；CuSO4°5H2O3.0g；KI44.5mg；MnSO4°H2O1.5g；Na2MoO4°2H2O100mg；H3BO310mg；FeSO4°7H2O32.5g；ZnCl210g；蒸馏水至500mL)补充基础盐培养基。
通过三步过程获得微生物的生长和异源蛋白质的表达。约24小时的第一批生长阶段(甘油作为碳源)，接着是约4小时的补料分批阶段(甘油作为限制性碳源)。在首先的这两个阶段期间，通过加入20％NH4OH将pH维持在5.0。一旦全部甘油被消耗，起始甲醇补料以引发eα’蛋白表达并且通过加入20％NH4OH将pH改变至6.0。甲醇补料分批阶段持续约24小时。在这两个补料分批阶段，通过搅拌器速度(rpm)的精密电子控制，而手动渐进地提高通气速率(vvm)将溶解氧(DissolvedOxygen)(DO％)稳定维持在30％。每几个小时，通过跟随DO％水平检查碳源限制性环境：在突然停止甲醇补料后它的数据应该显示急剧增加，并且反之亦然，在恢复甲醇之后迅速降低。每几个小时，从发酵罐中在无菌环境下取得样品，用于下述分析：光密度(λ600nm)、细胞生物质％(湿重)、无菌、显微镜观察、SDS-PAGE。
eα’纯化：发酵肉汤的下游加工。将约9.2L培养物从发酵罐中流出并且在冰上冷却。在+4℃实施全部随后的操作。通过在3,000×g达30分钟离心(CentrikonT-124，KontronInstruments)分离全部培养物；丢弃生物质(片状沉淀物)；通过在Zetaplus30SP(Cuno)上深度过滤澄清约7.5L上清液，并且随后在0.22μm过滤器(Millipak100，Millipore)上微过滤上清液。通过在MWCO10kDa的聚醚砜膜(Omegafilter，PalD上超滤浓缩澄清的滤液。浓缩物，约300mL，对3.0LTris-HCl10mMpH7.2渗滤并最终被低压冻干。用这个流程获得34.5g冻干粉，在SDS-PAGE上显示了相当低程度的污染物蛋白质并且具有约25％p/p的总蛋白质含量(BradfordProteinAssay，牛血清白蛋白作为校准标准)。
eα’纯化：色谱纯化。
为了更高性能的凝胶电泳，根据提供者的流程使用Pre-CastGelSystem(Invitrogen)。将两克冻干粉溶解于150mL50mMTris-HCl，pH7.50中并且将其装载到用相同的缓冲液平衡的DEAE-纤维素柱上(6×10cm，Whatman，Maidstone，UK)。用分别含有150和250mMNaCl的相同缓冲液实施保留的蛋白质的洗脱。用0.25MNaCl(300mL)洗脱的级分展示了eα’的最大含量。通过冷冻-干燥将溶液浓缩至100mL，并且随后用6000-8000Da膜在4℃用milliQ超滤水透析溶液24小时并随后冷冻干燥。获得约370mg蛋白质。
为了验证蛋白的均质性，将约1mg蛋白质装载到对称C4(4.6×250mm)反相柱上。使用缓冲液A(超滤水和0.1％三氟乙酸)和缓冲液B(100％乙腈+0.1％三氟乙酸)。
电泳技术。在12％聚丙烯酰胺凝胶上，根据参考文献11，使用mini-ProteanIIcell(Bio-Rad)实施在还原条件下(2％β-巯基乙醇)的SDS-PAGE。用考马斯蓝(CoomassieBlue)给凝胶染色。
细胞培养物。建立的人肝细胞瘤细胞系(HepG2)获取自美国典型培养物保藏中心(AmericanTypeCultureCollection)(Rockville，MD)。Eagle极限必需培养基(MEM)、胎牛血清、胰蛋白酶-EDTA(1x)、青霉素(105U/L)、链霉素(100g/L)、tricine缓冲液(1mmol/L，pH7.4)和非必需氨基酸溶液(100x)来自GIBCO(Madison，WI)。培养皿来自COSTAR(Cambridge，MA)。过滤器来自Millipore(Bedford，MA)。ProteinCoomassiePlusProteinAssay试剂盒购自Pierce(Rockford，IL，美国)。100mmol/LNaOH中无载体的碘125来自PerkinElmerLifeSciences(Boston，MA)。SephadexG25柱(PD10)来自PharmaciaBiotech(Uppsala，瑞典)。LDH和MTT试剂盒来自SigmaDiagnostics(Milano-意大利)。其他分析级的化学品来自Merck(Darmstadt，Germany)。在90mm直径的培养皿中单层种植细胞，并且维持在37℃、95％空气，5％CO2的湿化的大气和用10％胎牛血清(FCS)、非必需氨基酸溶液(1％，v/v)、青霉素(105U/L)、链霉素(0.1g/L)、tricine缓冲液(20mmol/L，pH7.4)、NaHCO3(24mmol/L)和丙酮酸钠(0.11g/L)补充的MEM中。对于设计用来评价LDL受体调节的实验，将细胞种在35mm塑料盘中(3-5x105个细胞)并且在将要达到汇合之前使用。在全部细胞培养物实验中，每2-3天更换培养基。为了评价细胞活力，基本如参考文献9中所描述的，通过甲基四唑盐(MTT)测定检测来自暴露于不同浓度eα’的细胞的培养基。通过使用动态(LDH/LD)诊断试剂盒(SigmaDiagnostics)测量乳酸脱氢酶(LDH)活性确定细胞酶渗漏。通过顺序制备超离心[12]从临床健康的正常血脂的志愿者的血浆中分离LDL(1.019≤d≤1.063g/L)。根据如由Bilheimer等人修改的McFarlane方法[13]，和先前描述的[3]方法标记脂蛋白。125I-LDl通过过滤(Millipore过滤器，0.45μm孔径)灭菌并且在4℃贮藏直到使用。如先前描述地制备人脂蛋白缺陷型血清(LPDS)[9]。
125I-LDL的摄取和降解。在含有/缺乏列于表中的不同浓度的eα’或3.5μmol/Lα’纯化的亚基或1.0μmol/L辛伐他汀(simvastatin)的情况下，在37℃用5g/100gLPDS补充的MEM中预先孵育单层细胞24小时以增量调节LDL-受体[2]。随后向培养基中加入固定浓度(7.5mg/L)的125I-LDL并且在37℃继续孵育另外5小时。如先前报道地评价125I-LDL的特定的摄取(结合+内化)和降解[2]。
统计分析。通过ANAOVA以及随后的Dunnett检测确定在用不同浓度的eα’孵育细胞之后LDL细胞摄取和降解的差异。以平均值±SD表示数值；认为p值＜0.05为统计显著的。
结果
巴斯德毕赤酵母中eα’的表达。在图1中显示了用于转化巴斯德毕赤酵母细胞的质粒的结构。在图2中显示了插入片段的序列和它与α’亚基的比对。如在材料和方法中所提到的，重组和野生型多肽之间仅存的差异在于N-端第一个氨基酸残基，由于技术原因，所述氨基酸残基在重组链中为丙氨酸。如通过培养基的SDS-PAGE分析所判断的(未显示)，选择显示了最大量重组多肽生产的克隆用于大量生产。图3-A显示了在用甲醇接种之前(第1道)和1-8-19和25小时之后(第2、3、4、5道)酵母培养物上清液的电泳分析。如它所显示的，由于诱导，在约20kDa的表观分子量处出现明显的带。
eα’的纯化。通过色谱方法达到eα’的纯化。收集并通过SDS-PAGE分析来自每一步的样品。在图3-B中显示了纯化步骤对鉴定的多肽的同质性的作用。在培养基中已经达到了非常低程度的由酵母蛋白质的污染，而其它的色谱步骤移除主要的污染物蛋白质并且以几乎同质的形式容许重组多肽回收。这条带的N-端序列分析证实了此20kDa多肽对应于eα’链。判断这一样品的纯度适合于细胞测定。
eα’的生物学活性。如下表所报道的，与未处理的细胞相比，向HepG2细胞加入作为阳性对照的纯化的α’亚基和它的截短的α’形式在LDL受体介导的摄取和降解中产生显著的上升。
表.α’和它的截短的形式(eα’)对HepG2细胞1，2的LDL摄取和降解上的作用
1数据为3个独立实验的平均值±SD，每一实验进行四次。＊P＜0.05对LPDS和＊＊P＜0.001对LPDS。
2在存在或缺乏不同浓度的重组多肽(eα’)或纯化的α’亚或辛伐他汀的情况下，在37℃具有5％LPDS的极限必需培养基中预孵育汇合的单层HepG2细胞达24小时。在加入125I-LDL(7.5mg/L培养基)之后，孵育细胞额外5小时。
4LPDS，脂蛋白缺陷型血清
结果显示了LDL调节是eα’剂量依赖性的并且最高浓度与阳性对照，辛伐他汀相似。如通过MTT和LDH测定所确定的(未显示)，任何浓度的eα’没有任何细胞毒性的证据。
根据本发明因而发现了能够诱导生物学应答的氨基酸序列存在于α’链的N-端延伸结构域。而且，我们发现N-端亲水片段在辛伐他汀的数量级级别的浓度下产生作用，所述辛伐他汀为强力的降血脂药。这个作用可至少部分地由于上述片段和硫氧还蛋白的体外相互作用，如本发明人所报道的[3]，所述硫氧还蛋白为在保守的活性位点序列Cys-Gly-Pro-Cys中具有氧还活性的二硫-二巯基化物的多功能性小蛋白质。这个发现可解释在用含有大豆蛋白的富含胆固醇的膳食饲养的兔中观察到的，相对于同样膳食但是含有酪蛋白作为蛋白质源饲养的兔中发现的，较长的由氧化铜诱导的LDL的氧化延迟期[22]。
获得的数据特别地有趣，因为它们首次显示了在体外模型中浓度小于10μM时大豆7S球蛋白α’链的N端亲水片段具有活性，所述浓度与那些辛伐他汀的报道类似。而且，重组蛋白的使用排除了其他蛋白质和非蛋白质大豆组分的任何牵连，所述其他蛋白质和非蛋白质大豆组分包括已经报道的缺少清晰益处并存在潜在毒性的异黄酮[23]。
本发明提供了对多种疾病具有有益作用的功能性的食品和组合物，所述疾病包括高脂血症和心血管疾病，所述食品或组合物在降脂疗法中单独使用或与药物组合使用，即他汀类药物如辛伐他汀、普伐他汀、氟伐他汀、阿托伐他汀、洛伐他汀。
使用常规的赋形剂和方法制备本发明的组合物。本发明的重组多肽的剂量将依赖于若干因素，比如患者重量、年龄和性别并且在多肽的药效学、药物动力学和毒理学特征的基础上由医生容易地确定。总之，然而，所述剂量为在约50到约500mg，一天一至三次范围内。
参考文献
1.SirtoriCR.等人，CurrAtherosclerRep.2001；3：47-53.
2.LovatiMR.等人，JNutr.1992；122：1971-8.
3.ManzoniC.等人，JNutr.2003；133：2149-55.
4.DurantiM.等人，JNutr.2004；134：1334-39.
5.Thanh，VH.等人，BiochimBiophysActa.1976；439：326-38.
6.MaruyamaN.等人，JAgricFoodChem.1999；47：5278-84.
7.SirtoriCR.等人，NutrMetabCardiovascDis.1998；8：334-40.
8.LovatiMR.等人，JAgricFoodChem.1998；46：2474-80.
9.LovatiMR.等人，JNutr.2000；130：2543-2549.
10.LovatiMR.等人.，FasebJ.2006；LB391：86.
11.LaemmliUK.Nature.1970；227：660-5.
12.HavelRY.等人，JClinInvest.1955；34：1345-53.
13.BilheimerDW.等人，.Biochim.Biophys.Acta1972；260：212-21.
14.SirtoriCR.等人，BritJNutr.2007；97：816-22.
15.LovatiMR.等人，NutrMetabCardiovascDis.1991；1：18-24.
16.LovatiMR.等人，JClinInvest.1987；80：1498-502.
17.KittsDD.等人，CurrPharmDes.2003；9：1309-23.
18.WanXS.等人，NutritionandCancer.2002；43：167-73.
19.ClementeA.等人，RecentProgressinMedicinalPlants.2008；20：397-417.
20.MatobaN.等人，FEBSLett.2001；497：50-4.
21.ChoSJ.等人，JAgricFoodChem.2008；56：4372-6.
22.CastiglioniS.等人，Atherosclerosis.2003；171：163-70.
23.SirtoriCR.等人，DrugSafety2001；24：665-82.
Claims (3)
1.大豆7S球蛋白α’亚基的N-端亲水片段，其氨基酸序列由SEQID1组成。
2.制备权利要求1的大豆7S球蛋白α’亚基的N-端亲水片段的方法，其包括所述N-端亲水片段的克隆、在巴斯德毕赤酵母中表达以及纯化。
3.包含与合适的载体混合的作为活性成分的权利要求1的大豆7S球蛋白α’亚基的N-端亲水片段的组合物。
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| EP09162939.4 | 2009-06-17 | ||
| PCT/EP2010/003627 WO2010145820A1 (en) | 2009-06-17 | 2010-06-16 | Cloning, yeast expression, purification and biological activity of the extension region of the soybean 7s globulin alfa' subunit involved in hep g2 cell cholesterol homeostasis |
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