一种GLP1-EGFa异源二聚体蛋白、功能及方法与流程

本发明涉及生物医药领域，尤其涉及一种glp1-egfa异源二聚体蛋白、功能及方法。
背景技术：
：高血糖是一种关于血糖的疾病，一般多发生于老年人中。高血糖的危害包括：1、导致机体脱水及高渗状态：高血糖致使大量葡萄糖随尿排泄，引起渗透性利尿，导致机体脱水，脱水使得细胞外液渗透压增高，水分由细胞内向细胞外转移引起细胞内失水，脑细胞失水可引起脑功能紊乱直至昏迷，临床上称之为“高渗性昏迷”；2、代谢紊乱：高血糖时，体内的蛋白质、脂肪均出现代谢紊乱，可引起各种并发症。急性并发症酮症酸中毒和高渗性昏迷。高渗性昏迷主要因血糖过高所致，有时可达500—600mg/d1，它可引起脑细胞和其它组织细胞脱水，导致昏迷；3、导致各种血管、神经慢性伴随症状：糖尿病病人长期高血糖会伤害血管及神经，导致心脑血管疾病、糖尿病肾病、视网膜病变、周围神经病变和糖尿病足坏死等慢性伴随症状的发生与发展；4、渗透压增高：高血糖时，细胞外液渗透压增高，细胞内液向细胞外流动，导致细胞内失水。当脑细胞失水时，可引起脑功能紊乱，临床上称高渗性昏迷。高血脂是一种常见的代谢异常，是指体血脂水平过高。高血脂的危害包括：1.心脑血管疾病：高血脂与高血压、高血糖同属于心血管事件的独立危险因素。长期血脂高，脂质在血管内皮沉积所引起的动脉粥样硬化，导致形成血栓阻塞血管，进而引起心脑血管疾病；2.急性胰腺炎：甘油三酯过高可能导致急性胰腺炎的发生。现有技术常用的降低血糖和血脂的方法包括：食疗、运动、药物等。glp-1由胰高血糖素原基因表达，在胰岛α细胞中，胰高血糖素原基因的主要表达产物是胰高血糖素，而在肠黏膜的l细胞中，前激素转换酶(pc1)将胰高血糖素原剪切为其羧基端的肽链序列，即glp-1。glp-1具有保护β细胞的作用glp-1可作用于胰岛β细胞,促进胰岛素基因的转录、胰岛素的合成和分泌,并可刺激胰岛β细胞的增殖和分化,抑制胰岛β细胞凋亡,增加胰岛β细胞数量。此外,glp-1还可作用于胰岛α细胞,强烈地抑制胰高血糖素的释放,并作用于胰岛δ细胞,促进生长抑素的分泌,生长抑素又可作为旁分泌激素参与抑制胰高血糖素的分泌。研究证明,glp-1可通过多种机制明显地改善2型糖尿病动物模型或患者的血糖情况,其中促进胰岛β细胞的再生和修复,增加胰岛β细胞数量的作用尤为显著,这为ⅱ型糖尿病的治疗提供了一个非常好的前景。前蛋白转化酶枯草杆菌蛋白酶kexin-9(pcsk9)是一种肝源性分泌蛋白，它与低密度脂蛋白受体(ldlr)的胞外区结合。pcsk9作为一种神经细胞凋亡调节转化酶，不但参与肝脏再生，调节神经细胞凋亡，还能通过降低肝细胞上ldlr的数量，影响ldl内化，使血液中ldl不能清除，从而导致高胆固醇血症。而来源于ldlr上的一个结构域(domain)的突变体egfa，可以特异性的结合pcsk9，因此可作为pcsk9抑制剂，能结合pcsk9并抑制循环型pcsk9再与ldlr的结合，从而阻止pcsk9介导的低密度脂蛋白受体降解，可以达到降血脂的效果。但是，现有技术的控制血糖、血脂的方法相对独立，没有一种高效的药物可以实现同时降血糖、降血脂以及长效的目的。技术实现要素：针对上述技术问题，本发明提供了一种可以长效的降血糖、降血脂的二聚体蛋白，且该二聚体蛋白获取可在体外复性重组获取。本发明的一个方面，提供了一种glp1-egfa异源二聚体蛋白，包括第一蛋白和第二蛋白；第一蛋白包括glp1或其变体，第二蛋白包括egfa或其变体。在另一优选例中，所述第一蛋白还包括第一fc变体，所述第二蛋白还包括第二fc变体，第一fc变体和第二fc变体采用knob-into-hole的方式连接。在另一优选例中，第一fc变体或第二fc变体的hole突变的氨基酸序列为seq_idno：16。seq_idno：16在另一优选例中，第一fc或第二fc的knob突变的氨基酸序列为seq_idno：17。seq_idno：17在另一优选例中，所述的第一蛋白还包括egfa或其变体，所述第二蛋白还包括glp1或其变体。在另一优选例中，glp1或其变体和egfa或其变体与第一fc变体或第二fc变体连接的连接肽包括：(gapq)n、(gepq)n和(ggggs)n，其中n为1-20。在另一优选例中，glp1-egfa异源二聚体蛋白的氨基酸序列选自seq_idno：1-15。seq_idno：1-15的模式图如图1所示。seq_idno：1seq_idno：2seq_idno：3seq_idno：4seq_idno：5seq_idno：6seq_idno：7seq_idno：8seq_idno：9seq_idno：10seq_idno：11seq_idno：12seq_idno：13seq_idno：14seq_idno：15在另一优选例中，glp1变体为glp1经过一个或多个氨基酸缺失突变所得的7-37个氨基酸的缩短形式，glp1变体的氨基酸序列选自seq_idno：18。seq_idno：18在另一优选例中，egfa变体为egfa经过一个或多个氨基酸缺失突变所得的314-353个氨基酸的缩短形式，egfa变体的氨基酸序列选自seq_idno：19。seq_idno：19本发明的第二个方面，提供了glp1-egfa异源二聚体蛋白的制备方法，包括，在足以表达权利要求1-10所述的glp1-egfa异源二聚体蛋白的条件下，培养权利要求13所述的宿主细胞，表达并纯化所述的glp1-egfa异源二聚体蛋白。在另一实施例中，方法包括，在足以表达glp1或其变体和egfa或其变体的条件下，培养表达glp1或其变体的宿主细胞和表达egfa或其变体的宿主细胞，将宿主细胞表达的glp1或其变体和egfa或其变体复性和纯化得到本申请所述glp1-egfa异源二聚体蛋白。在另一优选例中，所述的宿主细胞包括哺乳动物细胞。在另一优选例中，所述的宿主细胞包括hek293和cho。在另一实施例中，方法包括，在足以表达glp1或其变体和egfa或其变体的条件下，培养表达glp1或其变体的宿主细胞和表达egfa或其变体的大肠杆菌，将大肠杆菌表达的glp1或其变体和egfa或其变体复性和纯化得到本申请所述glp1-egfa异源二聚体蛋白。本发明还包括一种核酸，其编码本发明所述的glp1-egfa异源二聚体蛋白。本发明还包括一种dna载体，其包括编码本发明所述的glp1-egfa异源二聚体蛋白的核酸。本发明还包括一种宿主细胞，其转染有本发明所述的dna载体。本发明还包括一种药物组合物，其包含本发明所述的glp1-egfa异源二聚体蛋白、和可药用的赋形剂、稀释剂或载体。优选地，所述药用的赋形剂包括黏合剂、填充剂、崩解剂、润滑剂、助溶剂。本发明还包括一种靶向性蛋白分子，其包含本发明的glp1-egfa异源二聚体蛋白结构。所述glp1-egfa异源二聚体蛋白、所述的药物组合物、所述的靶向性蛋白分子，在制备用于治疗疾病或病症的药物中的用途。优选地，其中所述的疾病或病症为高血糖、糖尿病、以及有心血管疾病风险的糖尿病及并发症。优选地，其中所述的疾病或病症为高血脂、高脂血症。与现有技术相比，本发明的技术方案具有以下优点：1、本发明提供的二聚体蛋白兼具降低血糖和血脂的功能，并且长效；2、本发明的二聚体蛋白既可以在哺乳细胞表达，又可以由大肠杆菌表达的包涵体，经过变性和纯化获得；获取方式相对繁琐、复杂、昂贵的细胞表达具有更便捷、可控，以及具有成本优势；3、本发明采用优化的fc变体，实现了本发明的二聚体蛋白体外获取。附图说明为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。图1是本发明15种glp1-egfa异源二聚体蛋白和同源二聚体模式图。图2是基因692和基因693的pcr产物电泳图；图3是基因692和693蛋白表达电泳检测；图4是基因d(fc-egfa)的pcr产物电泳图；图5是基因d(fc-egfa)蛋白表达电泳检测结果；图6是source30q纯化蛋白电泳结果；图7是source15q纯化蛋白电泳结果；图8是source15q纯化蛋白sec-hplc检测结果；图9是3source15q纯化蛋白rp-hplc结果；图10是反复冻溶三次的rp-hplcoverlay结果；图11a是原始样品浓度rp-hplc结果；图11b是稀释蛋白浓度到0.06mg/ml的rp-hplc结果；图11c是稀释蛋白浓度到0.00006mg/ml的rp-hplc结果；图12是实验动物各组间不同周期血糖变化图；图13是实验动物各组间不同周期血清胆固醇(tc)变化图图14是实验动物各组间不同周期血清低密度脂蛋白(ldl)变化图；图15是实验动物各组间不同周期血清甘油三脂(tg)变化图；图16是实验动物各组间不同周期血清高密度脂蛋白(hdl)变化图；图17异二聚体复性非还原sds-page电泳图lane1，fc铰链区未突变；m，蛋白marker；lane2，fc铰链区带有efl233-235kae突变；图18是实施例3原样稀释方法图。具体实施方式以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。实施例1重组glp1-fc蛋白载体克隆和表达1.1、合成能够表达glp1(7-37)和higg4fc的基因(分别命名为基因a、基因b)以及相应的引物。能够表达glp1(7-37)的基因(基因a)的碱基序列为seq_idno：20能够表达higg4fc的基因(基因b)的碱基序列为seq_idno：21引物序列为：seq_idno：22-30名称序列692-f1-1gacgacgatgataaacacgcggaaggcaccttc692-f1-2taagaaggagatatacatatggacgacgatgataaacacgcg692-r1-1gcgcgccttgtggagcaccctgaggtgctccgccgcgacctctaaccagc692-r1-2accctgcggtgcaccttgcggcgcgccttgtggagca692-f2-1(xin)cgcaaggtgcaccgcagggtgctccgcagagttgtgcaccgaaggctg692-r2(xin)gtggtggtggtggtgctcgagttatttacccagactcaggctcag693-r1-1agcctccgccaccgctaccgccacctccgccgcgacctctaaccagc693-r1-2atccaccgccaccggagcctccgccaccgctac693-f2-1(xin)gaggcggaggttcaggtggaggtggcagcagttgtgcaccgaaggctga1.2、利用pcr和iionestepcloningkit(非连接酶依赖型单片段快速克隆试剂盒)对基因a和b进行点突变得到基因692，d4k-glp1(7-37)-(gapq)5-fc(228-447,del230p,efl233-235kae,t366w)；对基因a和b进行点突变得到基因693，d4k-glp1(7-37)-(g4s)5-fc(228-447,del230p,efl233-235kae,t366w)。基因692的碱基序列为：seq_idno：31基因693的碱基序列为：seq_idno：321.3、将基因692和基因693进行pcr扩增，并将扩增获得的pcr产物进行dna凝胶回收。pcr反应体系及条件为：pcr产物电泳结果如图2所示，基因692为单一的831bp条带，基因693为单一的846bp条带。1.4、将步骤1.3得到的2个基因片段进行重组反应反应体系：和反应条件为：37℃反应30min，立即置于冰上冷却，转化dh5α大肠杆菌。1.5、将步骤1.4的菌液进行pcr，pcr引物为：t7和t7t。并将pcr后的产物进行琼脂糖凝胶电泳进行检测。1.6、选择步骤1.3中的3个阳性克隆送生工进行测序。1.7、阳性菌液保存及质粒的提取。1.8、表达过夜培养的种子按1:50比例转接到含amp抗性的500mltb培养基的三角瓶中，初始od600大约为0.1，37℃，220rpm，培养至od600为2.5(大约3h),加入iptg(终浓度为0.5mmol/l)，37℃、220rpm过夜培养后收菌。1.9、蛋白电泳检测，将步骤1.8中表达的蛋白进行电泳检测，结果，如图3所示，图3左侧图为glp1-(gapq)5-fc的蛋白片段，图3右侧图为glp1-(g4s)5-fc的蛋白片段。实施例2重组fc-egfa蛋白载体克隆和表达2.1、合成能够表达egfa的基因(命名为基因c)以及相应的引物。能够表达egfa的基因(基因c)的碱基序列为seq_idno：332.2、利用pcr和iionestepcloningkit(非连接酶依赖型单片段快速克隆试剂盒)对基因b和c进行点突变和pcr得到基因d，fc(228-447,del230p,t366s/l368a/y407v)-(gepq)5-egfa。基因d的碱基序列为:seq_idno：342.3、将基因d进行pcr扩增，并将扩增获得的pcr产物进行dna凝胶回收。pcr反应体系及条件为：pcr产物电泳结果如图4所示，基因d为单一的840bp条带。2.4、将步骤2.3得到的基因片段进行重组反应反应体系：反应条件为：37℃反应30min，立即置于冰上冷却，转化dh5α大肠杆菌。2.5、将步骤2.4的菌液进行pcr，pcr引物为：t7和t7t。并将pcr后的产物进行琼脂糖凝胶电泳进行检测。2.6、选择步骤2.3中的4个阳性克隆送生工进行测序。2.7、将测序正确的阳性克隆接种于5mlamp抗性的lb液体培养基中，接种量为10μl。取500μl的菌液加入1.5ml的离心管中，加入500μl40％的甘油，用于保存。写好名称，宿主菌，日期，封上封口膜放入-80℃冰箱保存。剩余的菌液离心收集菌体，用于质粒提取。2.8、表达过夜培养的种子按1:50比例转接到含amp抗性的500mltb培养基的三角瓶中，初始od600大约为0.1，37℃，220rpm，培养至od600为2.5(大约3h)，加入iptg(终浓度为0.5mmol/l)，37℃、220rpm过夜培养后收菌。2.9、蛋白电泳检测，将步骤2.8中表达的蛋白进行电泳检测，结果，如图5所示。实施例3glp1-fc和fc-egfa异源二聚体的复性和纯化3.1溶解分别用尿素和dtt溶解实施例1和实施例2得到的glp1-(gapq)5-fc和fc-(gepq)5-egfa3.2复性按以上溶解的ib浓度，取glp1(gapq)-fc，25.8ml和fc-egfa，29ml按tp1:1混合，共696mg，搅拌30min，稀释到复性液中，室温过夜，作为复性样品，复性后的样品即为glp1-fc和fc-egfa异源二聚体。3.3以适当的比例在室温下ek酶切过夜，去除mdddk标签。3.4异源二聚体进行疏水作用层析phenyl纯化酶切后的样品加入适当的nacl后上70ml的phenyl柱子，然后进行nacl梯度洗脱，根据sds-page选择目的蛋白所在的收集管合并保存，此步可以捕获目的蛋白并去除一些杂蛋白。3.5异源二聚体进行离子交换层析source30q纯化疏水作用层析后合并的蛋白适当稀释后进行离子交换层析，采用nacl梯度洗脱，去除核酸和一些高聚蛋白以及杂蛋白。source30q纯化结果如图6所示，这是sds-page检测经过source30q纯化后收集起来的样品，非还原(nr)显示目的条带大约在60kd左右，为dimer的位置，还原电泳(r)在30kd左右。3.6异源二聚体进行离子交换层析source15q精细纯化离子交换层析source30q后合并的蛋白适当稀释后进行source15q精细纯化，采用nacl梯度洗脱，去除一些杂蛋白。source15q精细纯化结果如图7-9所示：图7为经过15q纯化后样品的sds-page；图8为sec-hplc分析纯化后的样品，结果显示为单一组分，不含聚合体和单体；图9为rp-hplc分析纯化后样品的纯度，大约在95％左右。3.7异源二聚体蛋白进行脱盐柱脱盐换液采用脱盐柱对异源二聚体蛋白进行脱盐换液处理，最终缓冲液为pbs,ph7.4。3.8内毒素检测采用鲎试剂法(lal)，测得内毒素为2.0eu/mg。3.9冻融及稀释稳定性实验3.9.1原样冻融结果操作：取20ul进行原样hplc检测；取100ul进行冻融：每次-80℃，30min，分别取30ul离心，反复三次，进行rp-hplc。hplc结果如图10所示，可见经过冻融三次之后，异源二聚体蛋白依然稳定。3.9.2按照图18所示的方式将原样进行稀释，将稀释后的样液进行rp-hplc，结果如图11a、11b、11c所示，峰形和保留时间相同，说明稀释后的蛋白依然稳定。实施例4异源二聚体蛋白在db/db小鼠的体内药效学初步研究4.1实验动物信息及饲养条件如下：动物种属小鼠性别male品系bsk-leprem2cd479/nju周龄或体重8-10周供应商江苏集萃药康生物科技有限公司动物到达日期2018/12/254.2实验方法1.所有动物首次称重为d-21，然后模拟给药期分别在d-14、d-10、d-3进行称重。2.所有动物从d-21至d-1天，每天上午(正式给药时间与之接近)进行皮下注射pbs(0.1ml/animal)。3.在d-3对所有动物进行血糖测定，同时采集血清进行tc、ldl、hdl、tg指标测定。4.d-3所有动物根据血糖和体重进行动物筛选和分组(血糖为主要指标，体重为次要指标)，分组时以笼为单位进行分组，每笼不足3只的进行微调，以免动物变换环境造成应激反应。5.给药周期为d0-d20，给药当天如果有体重、摄食量、血糖或血脂的测定时，应在操作完成后给药。6.给药开始后对组内所有动物，分别在不同时期进行体重、摄食量、血糖及血脂测定，并在实验终点收集血浆。7.在d21对所有动物进行体重、摄食量、血糖进行测定，并且采集血清进行tc、ldl、hdl、tg指标测定，同时采集血浆保存以待分析。4.3人道终点本实验在实施前，已取得iacuc委员批准通过。本实验中的动物操作及饲养依据aaalac2011版指南(internationalguidelinesasreportedintheguideforthecareanduseoflaboratoryanimals,nationalresearchcouncil2011)进行。在发生以下情况时(但不限于)，动物将执行安乐死，安乐死方式：吸入过量co2。a、无法摄食、严重脱水b、注射30分钟内，在出现立毛、眼睑下垂等不适时，继续观察30分钟，情况恶化c、不能站立(连续俯卧或者侧卧，无法站起)d、濒死，包括呼吸困难、衰竭动物体重下降超过20％时4.4实验结果初步研究表明，连续21天在db/db小鼠皮下注射30，100和300nmol/kg受试药glp-1/egfa或阳性对照liraglutide或lovastatin后，动物无明显临床症状。血糖水平：受试组glp-1/egfa与溶媒组相比，在不同剂量均有一定的降血糖作用；受试组与liraglutide组在给药后24小时血糖分别下降23.83、25.55、22.36和39.36％，且下降幅度持续到给药21天。与阳性对照liraglutide相比，受试药glp-1/egfa300nmol/kg与其降糖作用非常相似如图12。血脂水平如图13-16：受试组(glp-1/egfa)在连续给药1周后，除低剂量受试组外，对血清血清总胆固醇、低密度脂蛋白胆固醇及甘油三酯有显著的降低效果，且效果优于或等同于阳性对照lovastatin组。在高密度脂蛋白实验上，受试组(glp-1/egfa)与lovastatin组效果类似，均未见显著性差异。实施例5fc铰链区的氨基酸突变对异二聚体蛋白复性的影响采用实施例1和实施例2以及实施例3中3.1，3.2的方法，同时分别复性铰链区有efl233-235kae或fl234-235ak突变的以及没有突变的fc，sds-page显示，除了kih固有的突变外，铰链区有突变efl233-235kae对形成异二聚体有明显的增强作用，明显好于未带突变的(如图17所示)。以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本
技术领域：
中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。序列表<110>北京志道生物科技有限公司<120>一种glp1-egfa异源二聚体蛋白、功能及方法<160>34<170>siposequencelisting1.0<210>1<211>548<212>prt<213>人工序列(artificialsequence)<400>1sercysalaprolysalagluglyglyproservalpheleuphepro151015prolysprolysaspthrleumetileserargthrprogluvalthr202530cysvalvalvalaspvalserglngluaspprogluvalglnpheasn354045trptyrvalaspglyvalgluvalhisasnalalysthrlysproarg505560glugluglnpheasnserthrtyrargvalvalservalleuthrval65707580leuhisglnasptrpleuasnglylysglutyrlyscyslysvalser859095asnlysglyleuproserserileglulysthrileserlysalalys100105110glyglnproarggluproglnvaltyrthrleuproproserglnglu115120125glumetthrlysasnglnvalserleutrpcysleuvallysglyphe130135140tyrproseraspilealavalglutrpgluserasnglyglnproglu145150155160asnasntyrlysthrthrproprovalleuaspseraspglyserphe165170175pheleutyrserargleuthrvalasplysserargtrpglnglugly180185190asnvalphesercysservalmethisglualaleuhisasnhistyr195200205thrglnlysserleuserleuserleuglylysglyalaproglngly210215220alaproglnglyalaproglnglyalaproglnglyalaproglnhis225230235240alagluglythrphethrseraspvalsersertyrleugluglygln245250255alaalalysglupheilealatrpleuvalargglyargglysercys260265270alaproglupheleuglyglyproservalpheleupheproprolys275280285prolysaspthrleumetileserargthrprogluvalthrcysval290295300valvalaspvalserglngluaspprogluvalglnpheasntrptyr305310315320valaspglyvalgluvalhisasnalalysthrlysproarggluglu325330335glnpheasnserthrtyrargvalvalservalleuthrvalleuhis340345350glnasptrpleuasnglylysglutyrlyscyslysvalserasnlys355360365glyleuproserserileglulysthrileserlysalalysglygln370375380proarggluproglnvaltyrthrleuproproserglngluglumet385390395400thrlysasnglnvalserleusercysalavallysglyphetyrpro405410415seraspilealavalglutrpgluserasnglyglnprogluasnasn420425430tyrlysthrthrproprovalleuaspseraspglyserphepheleu435440445valserargleuthrvalasplysserargtrpglngluglyasnval450455460phesercysservalmethisglualaleuhisasnhistyrthrgln465470475480lysserleuserleuserleuglyglyalaproglnglyalaprogln485490495glyalaproglnglyalaproglnglyalaproglnglythrasnglu500505510cysleualaasnleuglyglycysserhisilecysarglysleuglu515520525ileglytyrglucysleucysproaspglypheglnleuvalalagln530535540argargcysglu545<210>2<211>549<212>prt<213>人工序列(artificialsequence)<400>2hisalagluglythrphethrseraspvalsersertyrleuglugly151015glnalaalalysglupheilealatrpleuvalargglyargglygly202530alaproglnglyalaproglnglyalaproglnglyalaproglngly354045alaproglnsercysalaprolysalagluglyglyproservalphe505560leupheproprolysprolysaspthrleumetileserargthrpro65707580gluvalthrcysvalvalvalaspvalserglngluaspprogluval859095glnpheasntrptyrvalaspglyvalgluvalhisasnalalysthr100105110lysproargglugluglnpheasnserthrtyrargvalvalserval115120125leuthrvalleuhisglnasptrpleuasnglylysglutyrlyscys130135140lysvalserasnlysglyleuproserserileglulysthrileser145150155160lysalalysglyglnproarggluproglnvaltyrthrleupropro165170175serglngluglumetthrlysasnglnvalserleutrpcysleuval180185190lysglyphetyrproseraspilealavalglutrpgluserasngly195200205glnprogluasnasntyrlysthrthrproprovalleuaspserasp210215220glyserphepheleutyrserargleuthrvalasplysserargtrp225230235240glngluglyasnvalphesercysservalmethisglualaleuhis245250255asnhistyrthrglnlysserleuserleuserleuglylysglythr260265270asnglucysleualaasnleuglyglycysserhisilecysarglys275280285leugluileglytyrglucysleucysproaspglypheglnleuval290295300alaglnargargcysgluglyalaproglnglyalaproglnglyala305310315320proglnglyalaproglnglyalaproglnsercysalaprogluphe325330335leuglyglyproservalpheleupheproprolysprolysaspthr340345350leumetileserargthrprogluvalthrcysvalvalvalaspval355360365serglngluaspprogluvalglnpheasntrptyrvalaspglyval370375380gluvalhisasnalalysthrlysproargglugluglnpheasnser385390395400thrtyrargvalvalservalleuthrvalleuhisglnasptrpleu405410415asnglylysglutyrlyscyslysvalserasnlysglyleuproser420425430serileglulysthrileserlysalalysglyglnproargglupro435440445glnvaltyrthrleuproproserglngluglumetthrlysasngln450455460valserleusercysalavallysglyphetyrproseraspileala465470475480valglutrpgluserasnglyglnprogluasnasntyrlysthrthr485490495proprovalleuaspseraspglyserphepheleuvalserargleu500505510thrvalasplysserargtrpglngluglyasnvalphesercysser515520525valmethisglualaleuhisasnhistyrthrglnlysserleuser530535540leuserleuglylys545<210>3<211>609<212>prt<213>人工序列(artificialsequence)<400>3hisalagluglythrphethrseraspvalsersertyrleuglugly151015glnalaalalysglupheilealatrpleuvalargglyargglygly202530alaproglnglyalaproglnglyalaproglnglyalaproglngly354045alaproglnsercysalaprolysalagluglyglyproservalphe505560leupheproprolysprolysaspthrleumetileserargthrpro65707580gluvalthrcysvalvalvalaspvalserglngluaspprogluval859095glnpheasntrptyrvalaspglyvalgluvalhisasnalalysthr100105110lysproargglugluglnpheasnserthrtyrargvalvalserval115120125leuthrvalleuhisglnasptrpleuasnglylysglutyrlyscys130135140lysvalserasnlysglyleuproserserileglulysthrileser145150155160lysalalysglyglnproarggluproglnvaltyrthrleupropro165170175serglngluglumetthrlysasnglnvalserleutrpcysleuval180185190lysglyphetyrproseraspilealavalglutrpgluserasngly195200205glnprogluasnasntyrlysthrthrproprovalleuaspser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tctgg420ttaaaggcttctacccgagcgatatcgcagttgagtgggaaagtaacggtcaaccggaga480acaactataaaaccaccccgccggttctggatagcgacggtagctttttcctgtacagtc540gtctgaccgttgataaaagccgttggcaggaaggcaacgtttttagctgtagcgtcatgc600acgaagccctgcataaccattacacccagaaaagcctgagcctgagtctgggtaaa656<210>22<211>33<212>dna<213>人工序列(artificialsequence)<400>22gacgacgatgataaacacgcggaaggcaccttc33<210>23<211>42<212>dna<213>人工序列(artificialsequence)<400>23taagaaggagatatacatatggacgacgatgataaacacgcg42<210>24<211>50<212>dna<213>人工序列(artificialsequence)<400>24gcgcgccttgtggagcaccctgaggtgctccgccgcgacctctaaccagc50<210>25<211>37<212>dna<213>人工序列(artificialsequence)<400>25accctgcggtgcaccttgcggcgcgccttgtggagca37<210>26<211>48<212>dna<213>人工序列(artificialsequence)<400>26cgcaaggtgcaccgcagggtgctccgcagagttgtgcaccgaaggctg48<210>27<211>45<212>dna<213>人工序列(artificialsequence)<400>27gtggtggtggtggtgctcgagttatttacccagactcaggctcag45<210>28<211>47<212>dna<213>人工序列(artificialsequence)<400>28agcctccgccaccgctaccgccacctccgccgcgacctctaaccagc47<210>29<211>33<212>dna<213>人工序列(artificialsequence)<400>29atccaccgccaccggagcctccgccaccgctac33<210>30<211>49<212>dna<213>人工序列(artificialsequence)<400>30gaggcggaggttcaggtggaggtggcagcagttgtgcaccgaaggctga49<210>31<211>825<212>dna<213>人工序列(artificialsequence)<400>31gacgacgatgataaacacgcggaaggcaccttcaccagcgatgtgagcagctatctggag60ggccaagcggctaaagaatttattgcgtggctggttagaggtcgcggcggagcacctcag120ggtgctccacaaggcgcgccgcaaggtgcaccgcagggtgctccgcagagttgtgcaccg180aaggaattcggcggtccgtctgtttttctgtttccgccgaaaccgaaagacaccctgatg240attagtcgtaccccggaagttacctgcgttgttgttgacgtcagccaggaagatccggaa300gttcagttcaactggtacgttgacggcgttgaagttcataacgcgaaaaccaaaccgcgc360gaagaacagttcaacagtacctatcgcgttgttagcgtactgaccgttctgcatcaggat420tggctgaacggcaaagagtacaaatgcaaagtcagcaacaaaggcctgccgagcagcatt480gaaaaaaccatcagcaaagcgaaaggccaaccgcgcgaaccgcaagtttataccctgccg540ccgtctcaggaagaaatgaccaaaaaccaggtcagcctgtggtgtctggttaaaggcttc600tacccgagcgatatcgcagttgagtgggaaagtaacggtcaaccggagaacaactataaa660accaccccgccggttctggatagcgacggtagctttttcctgtacagtcgtctgaccgtt720gataaaagccgttggcaggaaggcaacgtttttagctgtagcgtcatgcacgaagccctg780cataaccattacacccagaaaagcctgagcctgagtctgggtaaa825<210>32<211>840<212>dna<213>人工序列(artificialsequence)<400>32gacgacgatgataaacacgcggaaggcaccttcaccagcgatgtgagcagctatctggag60ggccaagcggctaaagaatttattgcgtggctggttagaggtcgcggcggaggtggcggt120agcggtggcggaggctccggtggcggtggatctggaggcggaggttcaggtggaggtggc180agcagttgtgcaccgaaggaattcggcggtccgtctgtttttctgtttccgccgaaaccg240aaagacaccctgatgattagtcgtaccccggaagttacctgcgttgttgttgacgtcagc300caggaagatccggaagttcagttcaactggtacgttgacggcgttgaagttcataacgcg360aaaaccaaaccgcgcgaagaacagttcaacagtacctatcgcgttgttagcgtactgacc420gttctgcatcaggattggctgaacggcaaagagtacaaatgcaaagtcagcaacaaaggc480ctgccgagcagcattgaaaaaaccatcagcaaagcgaaaggccaaccgcgcgaaccgcaa540gtttataccctgccgccgtctcaggaagaaatgaccaaaaaccaggtcagcctgtggtgt600ctggttaaaggcttctacccgagcgatatcgcagttgagtgggaaagtaacggtcaaccg660gagaacaactataaaaccaccccgccggttctggatagcgacggtagctttttcctgtac720agtcgtctgaccgttgataaaagccgttggcaggaaggcaacgtttttagctgtagcgtc780atgcacgaagccctgcataaccattacacccagaaaagcctgagcctgagtctgggtaaa840<210>33<211>120<212>dna<213>人工序列(artificialsequence)<400>33ggcaccaacgaatgtctggctaatctgggtggctgctctcacatctgtcgcaagctggaa60atcggctacgagtgtctgtgtccggacggctttcagctggtggcgcagcgtcgttgcgag120<210>34<211>834<212>dna<213>人工序列(artificialsequence)<400>34tcttgtgcaccggagttcctgggtggtccgagcgtgttcctgtttccaccgaaaccgaaa60gataccctgatgatttctcgtactccggaggtaacttgcgtagtagttgatgtgagccag120gaagatccggaggtgcagttcaactggtatgttgacggtgttgaggttcataacgccaag180actaaaccacgtgaagaacagttcaacagcacctaccgtgtggtaagcgtgctgaccgtt240ctgcaccaagattggctgaacggtaaggagtacaaatgtaaagtaagcaacaaaggcctg300ccgagcagcattgagaagaccatctctaaggctaaaggtcaaccacgcgagccgcaggtg360tacactctgccaccaagccaggaagagatgaccaagaatcaggtgtctctgagttgtgca420gttaagggtttctacccgagcgatattgcggtagaatgggaatctaacggtcagccggag480aacaactataagaccactccaccagttctggattccgacggctctttcttcctggtatcc540cgtctgaccgttgacaaatcccgttggcaggaaggcaacgtgttctcttgttctgttatg600cacgaggcgctgcacaaccactacacccagaagtctctgtccctgagcctgggtggagag660cctcagggtgaaccacaaggcgagccgcaaggtgaaccgcagggtgaaccgcagggcacc720aacgaatgtctggctaatctgggtggctgctctcacatctgtcgcaagctggaaatcggc780tacgagtgtctgtgtccggacggctttcagctggtggcgcagcgtcgttgcgag834当前第1页12