CN114644581B - 含芳基硫酚或芳基硒酚经修饰的氨基酸、重组蛋白及其生物合成方法及应用 - Google Patents
含芳基硫酚或芳基硒酚经修饰的氨基酸、重组蛋白及其生物合成方法及应用 Download PDFInfo
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Abstract
本发明提供含芳基硫酚或芳基硒酚经修饰的氨基酸、重组蛋白及其生物合成方法及应用。本发明基于大肠杆菌利用生物合成将几十种廉价易得的芳香或者脂肪巯基的前体小分子合成多种含芳基硫酚或芳基硒酚经修饰的氨基酸,并利用基因密码子扩展技术将上述氨基酸定点***到具有潜在应用的蛋白中,获得含有特定位点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白。其中***生物正交反应结构的上述氨基酸的蛋白可以偶联荧光分子,药物以及PEG,具有成像,治疗以及延长半衰期的潜在应用。
Description
技术领域
本发明属于生物医药领域,涉及非天然氨基酸、重组蛋白及其制备方法及应用,具体涉及含芳基硫酚或芳基硒酚经修饰的氨基酸、重组蛋白及其生物合成方法及应用。
背景技术
蛋白质不仅是细胞的组成成分之一,而且是细胞功能的主要执行者。自然界中蛋白质由20种天然氨基酸组成,这些天然氨基酸不同的理化性质赋予蛋白质结构和功能多样性。但是天然氨基酸因其结构类型的局限,限制了新型蛋白的开发。于是科学家在这20种天然氨基酸的基础上创造出能够编码蛋白质的新型非天然氨基酸(ncAAs)结构,极大丰富了蛋白质的结构与功能并且在化学生物学研究领域发挥着重要的作用。
基因密码子扩展技术便是其中之一,其能实现在生物体内任意目标蛋白的特定位点引入ncAAs。这一技术是利用与宿主细胞生物正交的氨酰tRNA合成酶(aminoacyl-tRNAsynthetase,aaRS)/tRNA对,在蛋白中突变的终止密码子(UAG/UGA/UAA)位点***ncAAs,得到定点编码非天然氨基酸的蛋白质,该技术可在蛋白的任意位点定点引入非天然氨基酸。
蛋白质药物如工程化抗体,重组蛋白等在治疗棘手的重大疾病和慢性疾病方面有重要应用,如肿瘤和免疫***等疾病。蛋白质化学修饰技术,在蛋白上修饰效应分子,赋予蛋白质新功能,可成为更强大的诊断工具或者治疗武器。例如,在蛋白质的特定氨基酸上化学标记荧光基团来追踪蛋白质在细胞内的定位和动态变化;通过在识别肿瘤的抗体上偶联药物分子,将药物靶向性和杀伤性有效的结合;在治疗性药物蛋白上偶联PEG,提高药物的半衰期等。蛋白质定点修饰可生产质量可控的蛋白修饰药物,而蛋白质定点修饰需要特定的生物技术来实现。基因密码子扩展技术可以将具有生物正交基团的非天然氨基酸定点编码到蛋白质的任意位置,是目前蛋白定点修饰的关键且成熟的技术。在蛋白质修饰上具有得天独厚的优势。
相对简单的ncAAs如酪氨酸或苯丙氨酸衍生物可以购买但是非常昂贵。而且大多数具有生物化学意义的ncAAs没有商业渠道购买,需通过多步合成来制备,有些ncAAs还需要进行手性拆分,产率较低,而且表达蛋白过程中对非天然氨基酸的需求量较大。这些因素限制了蛋白的大规模工业化生产制备。因此目前已有许多研究利用生物体(细菌,植物和动物)本身生物合成途径生产ncAAs或者通过引入市售廉价的前体分子生物合成ncAAs,并且运用上述基因密码子拓展技术***到目的蛋白产品中。
当前非天然氨基酸的生物合成已经取得了较好的应用成果,而且这些生物合成的非天然氨基酸也能通过基因密码子拓展技术或者营养缺陷手段也能同时***到蛋白质中。但是该技术却仍存在一些问题,比如:a.虽然找到了合成一些非天然氨基酸的生物途径但是大多数非天然氨基酸没有什么商业或者生物化学研究的价值;b.改造生成非天然氨基酸的生物途径较为复杂,而且生成氨基酸的产量较低,无法实现量产的目的;c.尽管体外加入了一些合成非天然氨基酸的前体分子,但是一些前体分子仍然需要化学合成,有些前体分子原料昂贵且合成产率低;d.一些生物途径的底物范围较为单一,不能生物合成一系列的非天然氨基酸类似物;e.尽管通过生物本身的合成途径生物合成出非天然氨基酸,但是***到蛋白上的产量较低,无利用价值。
发明内容
为了解决上述问题,本发明的目的在于提供一种含芳基硫酚或芳基硒酚经修饰的氨基酸,该氨基酸通过改造大肠杆菌的生物代谢途径进行表达,从而生物合成出一系列全新的含芳基硫酚或芳基硒酚经修饰的氨基酸。
本发明的另一个目的在于提供一种带有上述含芳基硫酚或芳基硒酚经修饰的氨基酸的残基的重组蛋白。
本发明的第三个目的在于提供一种偶联物。
本发明的第四目的在于提供上述含芳基硫酚或芳基硒酚经修饰的氨基酸的生物合成方法,该方法改造了大肠杆菌的生物代谢途径,通过添加芳香巯/硒基前体分子,表达出含芳基硫酚或芳基硒酚经修饰的氨基酸。
本发明的第五个目的在于提供一种制备定点***生物合成的含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的方法。
本发明的第六个目的在于提供上述偶联物的应用。
本发明的第五个目的在于提供使用上述重组蛋白与偶联剂偶联的偶联物。
为了实现上述目的,本发明提供一种含芳基硫酚或芳基硒酚经修饰的氨基酸,具有如下式(1)所示通式:
或其盐或其前药,其中,A为S或者Se,m和n相同或不同,m和n为0-3之间的整数;U为(C0-C4)直链或者支链烷基;
G取自叠氮基,乙酰基,四嗪基,2-氧代丙酰胺基团,硼酸基,醛基,氰基,羧基,三氟甲基或巯基;
含G官能团的键可以仅在苯环的邻位,间位或者对位,或者可同时存在于任意两个相同或不同位置;G1,G2和G3中的一个可以是-N-,或者-C-,且G1,G2和G3中的其余者独立的是-CH-。
其中,当G分别取自叠氮基,醛基,氰基,羧基时,U为C0烷基;
当G分别取自硼酸基,巯基时,m为0,同时U为C0烷基。
优选地,所述的含芳基硫酚或芳基硒酚经修饰的氨基酸,其中G为叠氮基,具有如下式(2)所示通式:
其中,m为0-3之间的整数;
叠氮基的键仅在苯环的邻位,间位或者对位,或者同时存在于任意两个相同或不同位置。
优选地,所述的含芳基硫酚或芳基硒酚经修饰的氨基酸,其中G为乙酰基,具有如下式(3)所示通式:
其中,m为0-3之间的整数;
乙酰基的键仅在苯环的邻位,间位或者对位,或者同时存在于任意两个相同或不同位置。
优选地,所述的含芳基硫酚或芳基硒酚经修饰的氨基酸,其中G为四嗪基,具有如下式(4)所示通式:
其中,m为0-3之间的整数;
四嗪基的键仅在苯环的邻位,间位或者对位,或者同时存在于任意两个相同或不同位置。
具体地,所述含芳基硫酚或芳基硒酚经修饰的氨基酸优选为如下式S-1至S-70或Se-1至Se-69中的任一式的化合物:
本发明还提供一种重组蛋白,所述重组蛋白包含如上述的含芳基硫酚或芳基硒酚经修饰的氨基酸的一个或多个残基。
本发明还提供一种偶联物,包含连接于偶联剂的如上述的重组蛋白,且所述偶联物任选地包含所述抗体和所述偶联剂之间的连接部分。
优选地,所述偶联剂为药物分子、荧光分子或PEG,连接部分为羟胺衍生物、DBCO衍生物、降冰片烯衍生物、BCN衍生物、环丙烯基团或反式环辛烯基团。
本发明还提供上述含芳基硫酚或芳基硒酚经修饰的氨基酸的生物合成方法,包括如下步骤:
1)构建带有半胱氨酸合成酶CysM基因和O-乙酰丝氨酸转移酶NtSat4基因或AtSat3基因的辅助质粒
a.使用带有持续性表达启动子Trc1序列的半胱氨酸合成酶CysM基因的引物从大肠杆菌基因组中扩增得到半胱氨酸合成酶CysM基因,使得该半胱氨酸合成酶CysM基因的前面融合Trc1启动子;将该半胱氨酸合成酶CysM基因通过Gibson组装方法克隆到含有p15a复制起点的大肠杆菌pBK表达载体中,该载体含有卡那霉素抗性基因,得到pBK-CysM质粒;
b.合成带有O-乙酰丝氨酸转移酶NtSat4基因的质粒或带有O-乙酰丝氨酸转移酶AtSat3基因的质粒,使用带有持续性表达启动子IPP序列的NtSat4基因的引物或带有持续性表达启动子IPP序列的AtSat3基因的引物从带有NtSat4或AtSat3基因的质粒中扩增得到带有持续性表达启动子IPP的NtSat4或AtSat3基因的片段;将该NtSat4基因或AtSat3基因的片段通过Gibson组装方式克隆到pBK-CysM载体中,得到辅助质粒pBK-CysM-NtSat4或pBK-CysM-AtSat3;
2)制备表达含芳基硫酚或芳基硒酚经修饰的氨基酸的菌株
将质粒pBK-CysM-NtSat4或质粒pBK-CysM-AtSat3转化入BL21(DE3)中,得到合成含芳基硫酚或芳基硒酚经修饰的氨基酸的菌株BL-pBK-CysM-NtSat4和BL-pBK-CysM-AtSat3;
3)含芳基硫酚或芳基硒酚经修饰的氨基酸的合成
将菌株BL-pBK-CysM-NtSat4或BL-pBK-CysM-AtSat3接种在5mL含有50μg/ml卡那霉素的2YT培养基中,过夜培养,次日按照接种量为1:100体积比转接于20mL含有50μg/ml卡那霉素的2YT培养基中,培养至对数生长初期,添加1mM芳香巯/硒基前体分子,在37℃,220转速的摇床中培养12小时;吸取1mL菌液,12000转速离心2分钟,吸取培养基上清,用0.22μm滤膜过滤,纯化。
优选地,步骤1)所述的带有持续性表达启动子Trc1序列的半胱氨酸合成酶CysM基因的引物由上游引物CysM-Trc1-F1、CysM-Trc1-F2、CysM-Trc1-F3和下游引物CysM-R组成;
其中,CysM-Trc1-F1的核苷酸序列如Seq ID No.1所示;CysM-Trc1-F2的核苷酸序列如Seq ID No.2所示;CysM-Trc1-F3的核苷酸序列如Seq ID No.3所示;CysM-R的核苷酸序列如Seq ID No.4所示;
含有p15a复制起点的大肠杆菌pBK表达载体的核苷酸序列如Seq ID No.5;
带有持续性表达启动子IPP序列的NtSat4基因的引物由上游引物NtSat4-F1、NtSat4-F2和下游引物NtSat4-R组成;
其中,NtSat4-F1的核苷酸序列如Seq ID No.11所示;NtSat4-F2的核苷酸序列如Seq ID No.12所示;NtSat4-R的核苷酸序列如Seq ID No.13所示;
带有持续性表达启动子IPP序列的AtSat3基因的引物由上游引物AtSat3-F1、AtSat3-F2和下游引物AtSat3-R组成;
其中,AtSat3-F1的核苷酸序列如Seq ID No.18所示;AtSat3-F2的核苷酸序列如Seq ID No.19所示;AtSat3-R的核苷酸序列如Seq ID No.20所示;
带有O-乙酰丝氨酸转移酶NtSat4基因的质粒的核苷酸序列如Seq ID No.10所示;NtSat4基因的核苷酸序列如Seq ID No.9所示;AtSat3基因的核苷酸序列如Seq ID No.17所示。
本发明还提供制备定点***生物合成含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的方法,包括如下步骤:
1)构建含有可***含芳基硫酚或芳基硒酚经修饰的氨基酸的包含氨酰tRNA合成酶基因和tRNA基因的辅助质粒pUltra-PhSeRS/tRNA
合成包含突变位点Y32W,L65H,H70G,F108N,Q109S,D158S和L162E的氨酰tRNA合成酶PhSeRS基因的质粒,将PhSeRS基因替换pUltra-CNF质粒中的CNF片段,其中pUltra-CNF质粒已包含tRNA基因序列,得到带有可***含芳基硫酚或芳基硒酚经修饰的氨基酸的辅助质粒pUltra-PhSeRS/tRNA;
2)构建含有终止密码子突变的表达目标蛋白的质粒
将C端带有6xHis标签的目标蛋白的基因序列用Gibson组装方法克隆到pET22b载体中,利用Quikchange定点突变的方法将特定位点的密码子突变为终止密码子TAG,得到带有TAG的目标蛋白的质粒;
3)表达具有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的大肠杆菌工程菌株
将前述制备的辅助质粒pBK-CysM-NtSat4或pBK-CysM-AtSat3、步骤1)制备的辅助质粒pUltra-PhSeRS/tRNA、步骤2)制备的带有TAG的目标蛋白的质粒共转化到大肠杆菌BL21(DE3)中,得到表达具有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的大肠杆菌工程菌株;
或,将步骤1)制备的辅助质粒pUltra-PhSeRS/tRNA、步骤2)制备的带有TAG的目标蛋白的质粒共转化到前述制备的菌株BL-pBK-CysM-NtSat4或BL-pBK-CysM-AtSat3中,得到表达具有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的大肠杆菌工程菌株;
上述两种方法均可得到得到表达具有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的大肠杆菌工程菌株,菌株为大肠杆菌BL21(DE3)中同时带有质粒pBK-CysM-NtSat4(或pBK-CysM-AtSat3)、pUltra-PhSeRS/tRNA、带有TAG的目标蛋白的质粒。
4)将所述大肠杆菌工程菌株培养至对数生长初期,添加1mM芳香巯/硒基前体分子,使用终浓度为0.5mM的IPTG诱导表达12-20小时,得到含有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白,然后用Ni-NTA Agarose进行纯化。
优选地,所述包含突变位点Y32W,L65H,H70G,F108N,Q109S,D158S和L162E的PhSeRS基因的质粒的核苷酸序列如Seq ID No.21所示。
优选地,所述大肠杆菌工程菌株培养的培养条件为:含有50μg/ml壮观霉素,100μg/ml氨苄霉素和50μg/ml卡那霉素的2-YT培养基在37℃条件下220rpm进行培养;对数生长初期为OD=0.3。
优选地,所述芳香巯/硒基前体分子为具有如式(5)的化学结构的通式化合物;
其中,A为SH或者SeH,m和n相同或不同,m和n为0-3之间的整数;U为(C0-C4)直链或者支链烷基;
G取自叠氮基,乙酰基,四嗪基,2-氧代丙酰胺基团,硼酸基,醛基,氰基,羧基,三氟甲基,巯基,硝基,羧甲基,胺乙酰基,卤素,氨基,甲氧基,羟基或卤素;
含G官能团的键可以仅在苯环的邻位,间位或者对位,或者可同时存在于任意两个相同或不同位置;G1,G2和G3中的一个可以是-N-,或者-C-,且G1,G2和G3中的其余者独立的是-CH-。
其中,当G分别取自叠氮基,醛基,氰基,羧基时,U为C0烷基;
当G分别取自硼酸基,巯基时,m为0,同时U为C0烷基。
优选地,所述卤素为-F、-Cl或-Br。
本发明还提供上述的重组蛋白与偶联物偶联后可作为荧光成像剂、治疗性药物以及延长半衰期药物应用。
本发明提供的重组蛋白由于将其中的天然氨基酸替换为非天然氨基酸(含芳基硫酚或芳基硒酚经修饰的氨基酸),而非天然氨基酸可人工设计为带有叠氮基团,乙酰基团,四嗪基团,二酮基团,硼酸基团,醛基团,苯胺基团等基团的非天然氨基酸,而这些基团为生物正交官能团,可以与连接有药物分子、荧光分子或PEG的偶联剂进行偶联,使得重组蛋白可以作为荧光成像剂、治疗性药物或延长半衰期药物使用。
本发明的有益效果在于:
本发明提供的含芳基硫酚或芳基硒酚经修饰的氨基酸、重组蛋白、含芳基硫酚或芳基硒酚经修饰的氨基酸的生物合成方法以及定点突变重组蛋白的制备方法,所使用的原料为可购买或者一步简单合成大量廉价易得的芳香以及脂肪巯基底物前体分子,而无需使用昂贵的非天然氨基酸;该方法不需要过度修改该生物合成的途径;可一次性生物合成以及定点***许多具有生物化学意义的含芳基硫酚或芳基硒酚经修饰的氨基酸到蛋白中;生成的具有新型结构的蛋白产量可以达到与未突变的野生型蛋白的产量的水平。
附图说明
图1为半胱氨酸衍生的含芳基硫酚或芳基硒酚经修饰的氨基酸的生物合成途径。
图2为半胱氨酸合成酶CysM基因上游融合Trc1启动子后的序列,以及引物位置关系图。
图3为本发明实施例所制备的pBK-CysM质粒的质粒图谱。
图4为本发明实施例所制备的pBK-CysM-NtSat4质粒的质粒图谱。
图5A至图5D为本发明实施例所制备的经修饰的氨基酸的LC-MS分析图谱。
图6为本发明实施例所制备的pUltra-PhSeRS质粒的质粒图谱。
图7为本发明实施例所制备的pET22b-sfGFP-Y151TAG质粒的质粒图谱。
图8A至图8N为本发明实施例所制备的BL-pET22b-sfGFP-Y151TAG菌株表达的***生物合成含芳基硫酚或芳基硒酚经修饰的氨基酸的sfGFP的质谱验证。
图9为本发明实施例所制备的pET22b-Herceptin-Fab质粒的质粒图谱。
图10为本发明实施例所制备***经修饰的氨基酸的Herceptin-Fab蛋白的质谱验证。
图11为定点***生物合成的经修饰的氨基酸的Herceptin-Fab蛋白可以与荧光物质、药物、PEG进行偶联的结构图。
图12为定点***生物合成的经修饰的氨基酸的Herceptin-Fab蛋白的偶联PEG或荧光基团的原理示意图。
图13***苯基叠氮半胱氨酸的Herceptin-Fab与DBCO-PEG20K偶联反应后的SDS-PAGE电泳结果图。
图14***苯基叠氮半胱氨酸的Herceptin-Fab与DBCO-Cy5偶联反应后用于HER2阳性肿瘤细胞的成像图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下文中将对本发明的实施例进行详细说明。需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互任意组合。本发明的目的和其他优点可通过在说明书、权利要求书以及附图中所特别指出的结构来实现和获得。
在本发明中,采用的基因密码子扩展技术可以参见Peter Schultz于2001年提出的在蛋白质中***人工合成的非天然氨基酸的技术(Science 2001,Vol 292,498-500。自然界中所有的蛋白都是由61个密码子编码20种氨基酸组成,其中TAG、TAA和TGA三个终止密码子执行蛋白翻译的终止。该技术的关键则是改变TGA及TAG的生物功能,将TGA及TAG变成编码非天然氨基酸的密码子。将人工合成的具有特殊物理和化学特性的“人造氨基酸”***到任何选定蛋白的任何选定位点。借住这些人造氨基酸可以将功能各异、具有特殊化学、物理或者生物活性官能团定点引入到蛋白质之中,例如,羰基、炔基和叠氮基团等特殊化学基团,这些基团能够有效地且选择性地形成共价键,更加有利于蛋白质的定点特异性修饰,从而改善蛋白的性质或者增加蛋白质的功能。到目前为止,该技术已经成功的将几十种非天然氨基酸定点表达在活细胞蛋白质中,赋予了这些蛋白质新的物理、化学、生理性质。这种定点修饰既不同于随机修饰的无质控标准,也不同于上述三种定点修饰,需要在被修饰蛋白上添加特定氨基酸序列。
关于“生物正交反应”的概念参考:Hang,H.C.,Yu,C.,Kato,D.L.,and Bertozzi,C.R.(2003).A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation.Proceedings of the National Academy of Sciencesof the United States of America 100,14846-14851。该反应是指能在生物***中发生而且不干扰内源性生物化学过程的化学反应。相对于传统的标记方式,如荧光蛋白标记,以化学小分子作为标记物好处在于他们更容易进入细胞,且具备高选择性。生物正交反应使得对生物体内的生物分子(如糖类、蛋白质和脂类)的实时研究成为可能。经过十多年的努力,一系列生物正交反应被广泛应用于细胞标记、抗体修饰、高通量测序、蛋白质组学等领域。目前已发展了大量满足生物正交性的化学偶联策略,如叠氮化合物与环炔烃的1,3-偶极环加成反应(又称无铜点击化学)(Baskin,J.M.,Prescher,J.A.,Laughlin,S.T.,Agard,N.J.,Chang,P.V.,Miller,I.A.,Lo,A.,Codelli,J.A.,and Bertozzi,C.R.(2007).Copper-free click chemistry for dynamic in vivo imaging.Proceedings oftheNational Academy of Sciences 104,16793-16797.)、硝酮与环炔烃的反应(Ning,X.,Temming,R.P.,Dommerholt,J.,Guo,J.,Ania,D.B.,Debets,M.F.,Wolfert,M.A.,Boons,G.-J.,and van Delft,F.L.(2010).Protein Modification by Strain-PromotedAlkyne-Nitrone Cycloaddition.Angewandte Chemie International Edition 49,3065-3068.)、醛或酮形成肟或腙的反应、四嗪与环状烯烃或环状炔烃的狄尔斯-阿尔德反应(Blackman,M.L.,Royzen,M.,and Fox,J.M.(2008).Tetrazine Ligation:FastBioconjugation Based on Inverse-Electron-Demand Diels-AlderReactivity.Journal of the American Chemical Society 130,13518-13519.)、基于异氰化物的点击反应(H.,Neves,A.A.,Stairs,S.,Brindle,K.M.,and Leeper,F.J.(2011).Exploring isonitrile-based click chemistry for ligationwithbiomolecules.Organic&biomolecular chemistry 9.),以及四环烷偶联反应(Sletten,E.M.,and Bertozzi,C.R.(2011).A Bioorthogonal QuadricyclaneLigation.Journal of the American Chemical Society 133,17570-17573.)。
本发明所提供的方法只需要购买一些廉价易得的商业化产品,或者一些仅需要在实验室通过简单的一两步合成即可得到的芳香巯/硒基前体分子,并将这些前体小分子在工程大肠杆菌内生成非天然氨基酸,并且通过基因密码子拓展技术将非天然氨基酸***到目的蛋白上。本发明提供的大肠杆菌工程菌株及重组蛋白的制备方法大大的降低了生产成本,极具工业应用潜力。
本发明得到的含有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白,所包含的非天然氨基酸残基结构如式(6)所示:
其中,所述Q为具有如式(7)所述通式结构;
其中,A为S或者Se,m和n相同或不同,m和n为0-3之间的整数;U为(C0-C4)直链或者支链烷基;
G取自叠氮基,乙酰基,四嗪基,2-氧代丙酰胺基团,硼酸基,醛基,氰基,羧基,三氟甲基,巯基,硝基,羧甲基,胺乙酰基,卤素,氨基,甲氧基,羟基或卤素;
含G官能团的键可以仅在苯环的邻位,间位或者对位,或者可同时存在于任意两个相同或不同位置;G1,G2和G3中的一个可以是-N-,或者-C-,且G1,G2和G3中的其余者独立的是-CH-。
其中,当G分别取自叠氮基,醛基,氰基,羧基时,U为C0烷基;
当G分别取自硼酸基,巯基时,m为0,同时U为C0烷基。
所述卤素为-F、-Cl或-Br。
在本发明下述具体实施例中非天然氨基酸特指含芳基硫酚或芳基硒酚经修饰的氨基酸,在部分表述中也简化为经修饰的氨基酸。
本发明的重组蛋白与已有的蛋白(如治疗蛋白、诊断蛋白、工业用酶,或其片段等)至少有60%、70%、75%、80%、90%、95%、99%或更高的相同性,其中包含一个或多个非天然氨基酸。可被修饰使其包含一个或多个非天然氨基酸的治疗蛋白、诊断蛋白以及其它蛋白包括但不限于α-1抗胰蛋白酶、血管抑素、抗溶血因子、抗体、载脂蛋白、脱辅基蛋白、心房利钠因子、心房利钠多肽、心房肽、C-X-C趋化因子、T39765、NAP-2、ENA-78、gro-a、gro-b、gro-c、IP-10、GCP-2、NAP-4、SDF-1、PF4、MIG、降钙素、C-kit配体、细胞因子、CC趋化因子、单核细胞趋化蛋白-1、单核细胞趋化蛋白2、单核细胞趋化蛋白-3、单核细胞炎性蛋白-1α、单核细胞炎性蛋白-1β、RANTES、I309、R83915、R91733、HCC1、T58847、D31065、T64262、CD40、CD40配体、C-kit配体、胶原蛋白、集落刺激因子(CSF)、补体因子5a、补体抑制剂、补体受体1、细胞因子、上皮中性粒细胞活化肽-78、MIP-16、MIP-1、表皮生长因子(EGF)、上皮中性粒细胞活化肽、***(EPO)、去角质毒素、因子IX、因子VII、因子VIII、因子X、成纤维细胞生长因子(FGF)、纤维蛋白原、纤连蛋白、四螺旋束蛋白、G-CSF、glp-1、GM-CSF、葡糖脑苷脂酶、***、生长因子、生长因子受体、刺猬蛋白、血红蛋白、肝细胞生长因子(hGF)、水蛭素、人体生长激素(hGH)、人血清白蛋白、ICAM-1、ICAM-1受体、LFA-1、LFA-1受体、胰岛素、***(IGF)、IGFI、IGF-II、干扰素(IFN)、IFN-α、IFN-β、IFN-γ、白介素(IL)、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、角质细胞生长因子(KGF)、乳铁蛋白、白血病抑制因子、荧光素酶、神经生长因子(neurturin)、中性粒细胞抑制因子(NIF)、制瘤素M、成骨蛋白、癌基因产物、paracitonin、甲状旁腺激素、PD-ECSF、PDGF、肽激素、多效生长因子(pleiotropin)、蛋白A、蛋白G、pth、致热外毒素A、致热外毒素B和致热外毒素C、pyy、松弛素、肾素、SCF、小的生物合成的蛋白质、可溶性补体受体I、可溶性I-CAM 1、可溶性白介素受体、可溶性TNF受体、生长调节素、生长抑素、生长激素、链激酶、超抗原、葡萄球菌肠毒素、SEA、SEB、SECT、SEC2、SEC3、SED、SEE、类固醇激素受体、超氧化物歧化酶、中毒性休克综合征毒素、胸腺素α1、组织型纤溶酶原激活物、肿瘤生长因子(TGF)、肿瘤坏死因子、肿瘤坏死因子α、肿瘤坏死因子β、肿瘤坏死因子受体(TNFR)、VLA-4蛋白、VCAM-1蛋白、血管内皮生长因子(VEGF)、尿激酶、mos、ras、raf、met、p53、tat、fos、myc、jun、myb、rel、***受体、孕激素受体、***受体、醛固酮受体、LDL受体、和皮质酮等。在相关的或进一步的实施方案中,所述经修饰的氨基酸多肽也可以与生长激素超基因家族的任何多肽成员是同源的。
如图1所示为生物合成含芳基硫酚或芳基硒酚经修饰的氨基酸(以半胱氨酸类似物为例)的反应原理示意图,其中,整条路线是基于大肠杆菌中半胱氨酸的生物合成途径,在生成半胱氨酸的最后一步是由半胱氨酸合成酶(简称:CysM酶)以乙酰基丝氨酸和硫氢根离子为底物,催化生成半胱氨酸。CysM酶除了识别硫氢根离子以外,还可以识别不同结构的带有巯基或硒基的有机小分子。本发明利用CysM酶的这种底物识别广泛的特点,人为添加芳香或者脂肪巯基/硒基小分子,最终生成结构类似于半胱氨酸的非天然氨基酸。为了进一步优化生物合成途径,提高非天然氨基酸产量,通过表达不受半胱氨酸反馈抑制的上游关键酶O-乙酰丝氨酸转移酶基因AtSat3或NtSat4,进一步提高非天然氨基酸产量。
所述芳香或者脂肪巯基/硒氢基小分子为具有如式(5);
其中,A为SH或者SeH,m和n相同或不同,m和n为0-3之间的整数;U为(C0-C4)直链或者支链烷基;
G取自叠氮基,乙酰基,四嗪基,2-氧代丙酰胺基团,硼酸基,醛基,氰基,羧基,三氟甲基,巯基,硝基,羧甲基,胺乙酰基,卤素,氨基,甲氧基,羟基或卤素;
含G官能团的键可以仅在苯环的邻位,间位或者对位,或者可同时存在于任意两个相同或不同位置;G1,G2和G3中的一个可以是-N-,或者-C-,且G1,G2和G3中的其余者独立的是-CH-。
其中,当G分别取自叠氮基,醛基,氰基,羧基时,U为C0烷基;
当G分别取自硼酸基,巯基时,m为0,同时U为C0烷基。
优选地,所述卤素为-F、-Cl或-Br。
优选符合上述式(2)的具体的芳香或者脂肪巯基/硒基小分子,用于下述具体实施例。
其中化合物1为化合物2通过生物合成的非天然氨基酸的形式,化合物2至化合物56为优选的芳香或者脂肪巯基/硒基小分子,在大肠杆菌工程菌株内通过生物合成非天然氨基酸,并通过基因密码子扩展技术定点的***到目标蛋白质中。
所用的的O-乙酰丝氨酸转移酶编码自烟草的NtSat4基因或拟南芥的AtSat3基因,均对半胱氨酸不敏感。其中,AtSat3基因编码的O-乙酰丝氨酸转移酶为线粒体酶,N端线粒体信号肽已删除。上述两个基因采用基因合成的方法,在持续性表达IPP启动子下和CysM的共表达。
试剂:
KOD OneTM PCR Master Mix(TOYOBO),DpnI(New England Biolabs,NEB),壮观霉素(Sigma),卡那霉素(Sigma),氨苄霉素(Sigma),1×PBS(Macgene),Ni亲和柱(GEhealthcare),IPTG(INALC),2-YT培养基(16g tryptone,12g yeast extract,5g NaCl,OXOID),pUltra-CNF表达载体(Addgene Plasmid No.48215),Gibson组装预混液(NEB),质粒提取试剂盒(天根质粒小提试剂盒),LB平板(10g/L蛋白胨,5g/L酵母提取物,10g/LNaCl,15g/L琼脂)。
基因序列合成、引物合成、基因测序均由金斯瑞生物科技股份有限公司完成。
大肠杆菌BL21(DE3)、大肠杆菌DH5α、大肠杆菌DH10B均为常规商业菌株。
需要说明的是,本发明中未详述的实验操作均为分子生物学常规操作。
实施例1:芳香巯基/硒基前体分子的获得
本发明所提供的芳香巯基/硒基前体分子属于廉价易得的,可直接进行购买的小分子,或者一些仅需要在实验室通过简单的一两步合成即可得到。这里以具体的含有叠氮以及苯乙酮基团的芳香巯基前体分子,为例展示的一步合成法。
苯乙酮基化合物以对巯基苯乙酮(化合物7)为例,叠氮基化合物以对巯基叠氮(化合物22)为例进行一步法合成。
A.对巯基苯乙酮(1-(4-mercaptophenyl)ethan-1-one,化合物7)的合成
对碘苯乙酮购买自毕得医药,产品编号BD11067。
CuSO4·5H2O购买自毕得医药,产品编号BD122189。
Cs2CO3购买自毕得医药,产品编号BD120462。
DMSO购买自海之园,产品编号124-10。
1,2-乙二硫醇购买自萨恩,产品编号A040973*。
乙酸乙酯购买自北京市通广精细化工公司。
无水硫酸钠购买自北京市通广精细化工公司。
硅胶购买自赛谱锐思(北京)科技有限公司,产品编号GJF200。
将原料对碘苯乙酮(1mmol),CuSO4·5H2O(12.5mg,0.05mmol),Cs2CO3(1.62g,5mmol),DMSO(2mL)。氩气保护下,加入1,2-乙二硫醇(0.18毫升,2mmol)。将混合物在110℃预热的油浴中搅拌20小时后冷却至室温,反应完毕加入20ml稀盐酸(5%),乙酸乙酯萃取两次(2×10ml),无水硫酸钠干燥过夜,真空浓缩,硅胶(200-300目)柱分离,得到灰色固体。负离子质谱验证m/z=151,证明合成产物确实为对巯基苯乙酮。A.对巯基苯乙酮(1-(4-mercaptophenyl)ethan-1-one,化合物7)的合成
对碘苯乙酮购买自毕得医药,产品编号BD11067。
CuSO4·5 H2O购买自毕得医药,产品编号BD122189。
Cs2CO3购买自毕得医药,产品编号BD120462。
DMSO购买自海之园,产品编号124-10。
1,2-乙二硫醇购买自萨恩,产品编号A040973*。
乙酸乙酯购买自北京市通广精细化工公司。
无水硫酸钠购买自北京市通广精细化工公司。
硅胶购买自赛谱锐思(北京)科技有限公司,产品编号GJF200。
将原料对碘苯乙酮(1mmol),CuSO4·5 H2O(12.5mg,0.05mmol),Cs2CO3(1.62g,5mmol),DMSO(2mL)。氩气保护下,加入1,2-乙二硫醇(0.18毫升,2mmol)。将混合物在110℃预热的油浴中搅拌20小时后冷却至室温,反应完毕加入20ml稀盐酸(5%),乙酸乙酯萃取两次(2×10ml),无水硫酸钠干燥过夜,真空浓缩,硅胶(200-300目)柱分离,得到灰色固体。负离子质谱验证m/z=151,证明合成产物确实为对巯基苯乙酮。
B.对巯基叠氮(4-azidobenzenethiol,化合物22)的合成
对氨基苯硫酚购买自北京偶合科技有限公司,货号01001091-5g。
叔丁基亚硝酸酯购买自杭州贝洛生物科技有限公司,货号IN-A17327-100ml。
叠氮基三甲基硅烷(TMSN3)购买自萨恩化学技术(上海)有限公司,产品编号W320011*。
硅胶购买自赛谱锐思(北京)科技有限公司,产品编号GJF200。
将原料对氨基苯硫酚(2g,15.97mmol)溶于乙腈(CH3CN)中,冰盐浴冷却至0℃,搅拌过程中滴加叔丁基亚硝酸酯(2.84ml,23.9mmol),继续搅拌10分钟,然后加入叠氮基三甲基硅烷(2.53ml,19.17mmol),氩气保护下室温反应1h,反应完毕,浓缩溶剂,硅胶(200-300目)柱分离(EP:EA=8:2)得到最终产品(收率80%)。负离子质谱验证m/z=150,证明合成产物确实为对巯基叠氮。
实施例2:构建带有半胱氨酸合成酶CysM基因和O-乙酰丝氨酸转移酶NtSat4基因或AtSat3基因的辅助质粒
由于烟草的NtSat4基因或拟南芥的AtSat3基因均可以应用于本发明,在本实施例中以烟草的NtSat4基因为例来构建辅助质粒。
1.使用带有Trc1序列的半胱氨酸合成酶CysM基因的引物从大肠杆菌BL21基因组中扩增得到半胱氨酸合成酶CysM基因,使得该半胱氨酸合成酶CysM基因的前面融合有持续性表达启动子Trc1;
其中,带有Trc1序列的半胱氨酸合成酶CysM基因的引物由上游引物CysM-Trc1-F1、CysM-Trc1-F2、CysM-Trc1-F3和下游引物CysM-R组成。
CysM-Trc1-F1的核苷酸序列如Seq ID No.1所示:GTCAGCGGTTAACAATTTCACACAGGAAACAGACCAAGGAGCCCATATGAGTACATTAGAACAAACAATAGGCAATACGC。
CysM-Trc1-F2的核苷酸序列如Seq ID No.2所示:TGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTCAGCGGTTAACAATTTCACAC。
CysM-Trc1-F3的核苷酸序列如Seq ID No.3所示:CGGGTTGTCAGCCTGTCCCGCTTATAAGATTGTTGACAATTAATCATCCGGCTC。
CysM-R的核苷酸序列如Seq ID No.4所示:CAATTTAGCGTTTGAAACTGCAGTCACTTGTCATCGTCGTCCTTGTAGTCAATCCCCGCCCCCTGGCTAAAA。
如图2所示为上游引物中融合有Trc1启动子序列Ptrc1的位置关系,通过菌落PCR反应将Ptrc1融合到CysM基因前面。在下游引物CysM-R上还设计有用于纯化蛋白的flag标签。
菌落PCR反应体系如表1所示:
表1菌落PCR反应体系
PCR反应条件为:95℃10min;98℃10s,54℃5s,68℃20s 30个循环;4℃保存。PCR产物经电泳,纯化,回收得到CysM基因片段。
2.将该半胱氨酸合成酶CysM基因通过Gibson组装方式(参考NatMethods.2009May;6(5):343-345)克隆到经改造的含有p15a复制起点的大肠杆菌pBK载体中得到pBK-CysM质粒。pBK载体序列如SeqID No.5所示。pBK载体在进行Gibson组装前进行PCR线性化。
pBK载体进行PCR线性化的具体过程为:利用pBK-F和pBK-R引物,以pBK载体为模板扩增。其中,pBK-F引物序列如Seq ID No.6所示:CTGCAGTTTCAAACGCTAAATTGC;pBK-R引物序列如Seq ID No.7所示:ATCTTATAAGCGGGACAGGCTG。PCR反应体系如表2所示。
表2 pBK载体线性化的PCR反应体系
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
pBK-F(10μM) | 2μL |
pBK-R(10μM) | 2μL |
pBK载体(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃30s,30个循环;4℃保存。PCR产物经电泳,纯化,回收得到线性化的pBK载体。
Gibson反应体系为:CysM基因片段(25ng/μL):1.5μL,线性化的pBK载体(50ng/μL):1μL,Gibson反应液:2.5μL,总体系为5μL。
Gibson反应条件为:50℃1小时。
Gibson反应液转化大肠杆菌DH10B,涂布于50μg/ml卡那霉素的LB平板,次日挑单菌落于5mL 2YT液体培养基,培养基中添加浓度为50μg/ml的卡那霉素,置于37℃,220转速摇床培养12小时,提质粒(按照试剂盒说明书操作),质粒经测序确认为pBK-CysM质粒。
如图3所示,为pBK-CysM质粒的质粒图谱,从图3可以看出pBK-CysM质粒以p15a为复制起点,具有卡那霉素抗性,以trc1为启动子表达CysM基因,在CysM基因末端还具有用于纯化蛋白的flag标签。pBK-CysM载体的序列如Seq ID No.8所示。
3.从文献Amino Acids 2003,24,195-203中得到NtSat4基因序列如Seq ID No.9所示,委托公司合成到质粒载体中,得到NtSat4基因合成质粒,序列如Seq ID No.10所示。
使用带有IPP序列的NtSat4基因的引物从NtSat4基因合成质粒中扩增得到O-乙酰丝氨酸转移酶NtSat4基因,使得该O-乙酰丝氨酸转移酶NtSat4基因的前面融合有持续性表达启动子IPP;
带有IPP序列的NtSat4基因的引物由上游引物NtSat4-F1、NtSat4-F2和下游引物NtSat4-R组成,其中,NtSat4-F1的核苷酸序列如Seq ID No.11所示:AAAAAAATATTCTCAACATAAAAAACTTTGTGTAATACTTGTACTCAAGGAGGATGGCTGCGGCGACCCCG;NtSat4-F2的核苷酸序列如Seq ID No.12所示:TAAATCGAACTTTTGCTGAGTTGAAGGATCAAAAAAATATTCTCAACATAAAAAACTTTGTGTAATAC;NtSat4-R的核苷酸序列如Seq ID No.13所示:AAGCGGGACAGGCTGACAACCCGAGGATCCAAAACGCCGCTGAACGCGGCGTTTTTTAAATAACATAATCGCTCCACTCGT。PCR反应体系如表3所示。
表3 PCR反应体系
ddH2O | 18μL |
KOD OneTM PCR Master Mix | 25μL |
NtSat4-F1(10μM) | 2μL |
NtSat4-F2(10μM) | 2μL |
NtSat4-R(10μM) | 2μL |
NtSat4基因合成质粒(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:95℃10min;98℃10s 54℃5s 68℃20s 30个循环;4℃保存。PCR产物经电泳,纯化,回收得到带有IPP启动子的NtSat4基因片段。
4.将该O-乙酰丝氨酸转移酶NtSat4基因通过Gibson组装方式克隆到pBK-CysM载体中,得到辅助质粒pBK-CysM-NtSat4。pBK-CysM质粒在进行Gibson组装前进行PCR线性化。
pBK-CysM质粒进行PCR线性化的具体过程为:利用pBK-CysM-F和pBK-CysM-R引物,以pBK-CysM质粒(Seq ID No.8)为模板扩增,pBK-CysM质粒线性化的PCR反应体系如表4所示。
其中,pBK-CysM-F引物的序列Seq ID No.14如所示:GGATCCTCGGGTTGTCAGC;pBK-CysM-R引物的序列Seq ID No.15如所示:GATCCTTCAACTCAGCAAAAGTTCG。
表4 pBK-CysM质粒线性化的PCR反应体系
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
pBK-CysM-F(10μM) | 2μL |
pBK-CysM-R(10μM) | 2μL |
pBK-CysM载体(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃40s,30个循环;4℃保存,经纯化得到线性化的pBK-CysM质粒。
Gibson反应体系为:NtSat4基因片段(20ng/μL):1.5μL,线性化的pBK-CysM(50ng/μL):1μL,Gibson反应液:2.5μL,总体系为5μL。
Gibson反应条件为:50℃1小时。
Gibson反应液转化大肠杆菌DH10B,涂布于50μg/ml卡那霉素的LB平板,次日挑单菌落于5mL 2YT液体培养基,培养基中添加浓度为50μg/ml的卡那霉素,置于37℃,220转速摇床培养12小时,提质粒(按照试剂盒说明书操作),质粒经测序确定为pBK-CysM-NtSat4质粒。
如图4所示为pBK-CysM-NtSat4质粒的质粒图谱,从图4可以看出,pBK-CysM-NtSat4质粒在卡那霉素抗性基因和CysM基因之间***了NtSat4基因。pBK-CysM-NtSat4质粒的序列如Seq ID No.16所示。pBK-CysM-NtSat4质粒在大肠杆菌中可以表达O-乙酰丝氨酸转移酶(NtSat4)和半胱氨酸合成酶(CysM),O-乙酰丝氨酸转移酶可将丝氨酸转化生成乙酰基丝氨酸,半胱氨酸合成酶可将乙酰基丝氨酸转化生成半胱氨酸类似物的非天然氨基酸。
当将辅助质粒中的NtSat4基因更换为Atsat3时,将从参考文献J Biol Chem1998,273,32739-32745中得到的如Seq ID No.17所示的Atsat3基因序列替代NtSat4基因,相应将上游引物NtSat4-F1、NtSat4-F2和下游引物NtSat4-R替换为AtSat3-F1、AtSat3-F2和AtSat3-R,可以制备得到pBK-CysM-Atsat3质粒;其中,AtSat3-F1的核苷酸序列如Seq IDNo.18所示:TCAAAAAAATATTCTCAACATAAAAAACTTTGTGTAATACTTGTACTCAAGGAGGATGGCGGCGTGCATTGAC;AtSat3-F2的核苷酸序列如Seq ID No.19所示:TAAATCGAACTTTTGCTGAGTTGAAGGATCAAAAAAATATTCTCAACATAAAAAACTTTGTGTAATAC;AtSat3-R的核苷酸序列如Seq ID No.20所示:AAGCGGGACAGGCTGACAACCCGAGGATCCAAAACGCCGCTGAACGCGGCGTTTTTTAGATCACGTAATCGCTCCATTC。
实施例3生物合成非天然氨基酸的工程菌株的制备
将质粒pBK-CysM-NtSat4或质粒pBK-CysM-AtSat3转化到BL21(DE3)感受态细胞中,转化按照《分子克隆实验指南(第四版)》的步骤进行,然后涂布于添加了50μg/ml卡那霉素的LB平板,次日挑取单菌落,在添加50μg/ml卡那霉素的2YT培养基中37℃,220rpm扩增培养12-16小时,后保种,得到合成非天然氨基酸的菌株BL-pBK-CysM-NtSat4或BL-pBK-CysM-AtSat3。
实施例4非天然氨基酸的合成与鉴定
将菌株BL-pBK-CysM-NtSat4接种在5mL含有50μg/ml卡那霉素的2YT培养基中,过夜培养,次日按照接种量为1:100体积比例转接于20mL含有50μg/ml卡那霉素的2YT培养基中,培养至对数生长初期,添加1mM化合物7或化合物55,在37℃,220转速的摇床中培养12小时。吸取1mL菌液,12000转速离心2分钟,吸取培养基上清,用0.22μm滤膜过滤,纯化(纯化过程参考Nat Biotechnol.2003Apr;21(4):422-7)。纯化产物进行LC-MS分析检测,结果如图5A至图5D所示。其中,图5A代表化合物7生成对应氨基酸的出峰时间,图5B表示化合物7出峰的物质的mass图。图5C代表化合物55生成对应氨基酸的出峰时间,图5D表示化合物55出峰的物质的mass图。
从图5A至图5D可以看出,本发明制备的菌株BL-pBK-CysM-NtSat4或BL-pBK-CysM-AtSat3确实可以生物合成非天然氨基酸,当根据需要添加不同的芳香或者脂肪巯基/硒基小分子可以得到具有相应结构的非天然氨基酸的产品。此外,也可以对菌株进行继续改造,使得非天然氨基酸在大肠杆菌体内直接合成目标重组蛋白。
实施例5辅助质粒pUltra-PhSeRS/tRNA的制备
根据参考文献:Angew Chem Int Ed Engl 2007,46,6849-6851得到突变位点为Y32W,L65H,H70G,F108N,Q109S,D158S和L162E的氨酰tRNA合成酶PhSeRS基因,并由公司合成带有突变位点的PhSeRS基因的质粒,序列如Seq ID No.21所示。将PhSeRS基因替换pUltra-CNF表达载体中的CNF片段,得到辅助质粒pUltra-PhSeRS/tRNA。
具体过程为:
1.利用PhSeRS-F,PhSeRS-R引物从带有PhSeRS基因的质粒中PCR扩增PhSeRS基因片段,其中,PhSeRS-F引物的序列如Seq ID No.22所示:CAAAGGAGGTGCGGCCGCATGGACGAATTTGAAATGATAAAGAGAAACAC;PhSeRS-R引物的序列如Seq ID No.23所示:ACCGTTTAAACGCGGCCGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATAAGTT。PCR反应体系如表5所示。
表5 PCR反应体系:
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
PhSeRS-F(10μM) | 2μL |
PhSeRS-R(10μM) | 2μL |
带有PhSeRS基因的质粒(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃20s,30个循环;4℃保存。PCR产物经电泳,纯化,回收得到PhSeRS基因片段。
2.然后将pUltra-CNF载体通过PCR方式线性化,通过Gibson组装方式克隆得到pUltra-PhSeRS/tRNA质粒。
pUltra-CNF载体进行PCR线性化的具体方法:利用pUltra-F和pUltra-R引物,以pUltra-CNF载体为模板扩增,pUltra-CNF载体线性化的PCR反应体系如表6所示。
其中,pUltra-F引物的序列如Seq ID No.24所示:GCGGCCGCGTTTAAACGG;pUltra-R引物的序列如Seq ID No.25所示:GCGGCCGCACCTCCTTTG。
表6 pUltra-CNF载体线性化的PCR反应体系
PCR反应条件为:98℃10s,54℃5s,68℃60s,30个循环;4℃保存,PCR产物经电泳,纯化,回收得到线性化的pUltra-CNF。
Gibson反应体系为:PhSeRS基因片段(20ng/μL):1.5μL,线性化的pUltra-CNF(50ng/μL):1μL,Gibson反应液:2.5μL,总体系为5μL。
Gibson反应条件为:50℃1小时。
Gibson反应液转化大肠杆菌DH10B,涂布于50μg/ml壮观霉素的LB平板,次日挑单菌落于5mL 2YT液体培养基,培养基中添加浓度为50μg/ml的壮观霉素,置于37℃,220转速摇床培养12小时,提质粒(按照试剂盒说明书操作),质粒经测序确认为pUltra-PhSeRS质粒。
如图6所示为pUltra-PhSeRS/tRNA质粒的质粒图谱,从图6可以看出,pUltra-PhSeRS/tRNA质粒具有壮观霉素抗性。pUltra-PhSeRS质粒序列如Seq ID No.26所示。
实施例6制备含突变sfGFP的质粒
通过基因合成获得序列如Seq ID No.27所示的带有sfGFP基因的质粒,将序列如Seq ID No.28所示的pET22b载体进行PCR线性化,sfGFP基因用Gibson组装方式克隆到pET22b载体中,得到pET22b-sfGFP质粒,利用quikchange定点突变的方式将sfGFP的151位Y突变为TAG,从而获得pET22b-sfGFP-Y151TAG质粒。
具体过程为:利用sfGFP-F,sfGFP-R引物从带有sfGFP基因的质粒中扩增sfGFP基因片段,其中,sfGFP-F引物的序列如Seq ID No.29所示:CACACAGAATTCATTAAAGAGGAGAAATTACATATGAGCAAAGGAGAAGAACTTTTCACT;sfGFP-R引物的序列如Seq ID No.30所示:GTCCAAGCTCAGCTAATTAAGCTTTTAGTGGTGGTGGTGGTGGTGGGATC,划线部分为6xHis标签。扩增sfGFP基因片段的PCR反应体系如表7所示。
表7扩增sfGFP基因片段的PCR反应体系
PCR反应条件为:98℃10s,54℃5s,68℃15s,30个循环;4℃保存,PCR产物经电泳,纯化,回收得到sfGFP基因片段。
将pET22b质粒通过PCR方式线性化,具体过程为:利用pET22b-sfGFP-F和pET22b-sfGFP-R引物,以pET22b质粒为模板扩增,pET22b质粒PCR反应体系如表8所示。
其中,pET22b-sfGFP-F引物序列如Seq ID No.31所示:AAGCTTAATTAGCTGAGCTTGGAC;pET22b-sfGFP-R引物序列如Seq ID No.32所示:ATGTAATTTCTCCTCTTTAATGAATTCTGTGTG。
表8 pET22b质粒PCR反应体系
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
pET22b-sfGFP-F(10μM) | 2μL |
pET22b-sfGFP-R(10μM) | 2μL |
pET22b质粒(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃60s,30个循环;4℃保存。PCR产物经电泳,纯化,回收得到线性化的pET22b质粒。
Gibson反应体系为:sfGFP基因片段(20ng/μL):1.5μL,线性化的pET22b质粒(50ng/μL):1μL,Gibson反应液:2.5μL,总体系为5μL。
Gibson反应条件为:50℃1小时。
Gibson反应液转化大肠杆菌DH10B,涂布于50μg/ml氨苄青霉素的LB平板,次日挑单菌落于5mL 2YT液体培养基,培养基中添加浓度为50μg/ml的氨苄青霉素,置于37℃,220转速摇床培养12小时,提质粒(按照试剂盒说明书操作),质粒经测序确认为pET22b-sfGFP质粒。测序得pET22b-sfGFP质粒序列如Seq ID No.33所示。
quikchange定点突变的步骤为:利用sfGFP-151-F,sfGFP-151-R引物,以pET22b-sfGFP质粒为模板进行PCR扩增,PCR产物利用DpnI内切酶酶切,酶切后的产物转化大肠杆菌,挑单克隆提质粒,测序验证得到pET22b-sfGFP-Y151TAG质粒。
其中,sfGFP-151-F引物的序列如Seq ID No.34所示:TTTAACTCACACAATGTATAGATCACGGCAGACAAACAAAAGAATG;sfGFP-151-R引物的序列如Seq ID No.35所示:GTTTGTCTGCCGTGATCTATACATTGTGTGAGTTAAAGTTGTACTCGAG;PCR反应体系如表9所示。
画横线的部分即为突变位点,将原编码酪氨酸(Y)的密码子TAC突变为终止密码子TAG。
表9PCR反应体系
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
sfGFP-151-F(10μM) | 2μL |
sfGFP-151-R(10μM) | 2μL |
pET22b-sfGFP质粒(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃80s,15个循环;4℃保存。
DpnI内切酶酶切反应体系为:50μL PCR反应液,1μL DpnI内切酶。
DpnI内切酶酶切反应条件为:37℃2小时。
大肠杆菌转化步骤为:将5μL DpnI酶切产物加入大肠杆菌DH10B化转感受态中,冰浴30min,42℃热激45秒,冰浴3分钟,加入2YT培养基37℃孵育1小时,涂板,挑单克隆,提取质粒,测序验证。
如图7所示为pET22b-sfGFP-Y151TAG质粒的质粒图谱,该pET22b-sfGFP-Y151TAG质粒具有氨苄青霉素抗性,测序确定该pET22b-sfGFP-Y151TAG质粒仅在Y151TAG位点与pET22b-sfGFP质粒有差别(TAC突变为TAG)。
实施例7.定点突变的sfGFP表达菌株的制备
将实施例2制备的辅助质粒pBK-CysM-NtSat4(卡那霉素抗性)、实施例5制备的pUltra-PhSeRS/tRNA质粒(壮观霉素抗性)和实施例6所获得的表达质粒pET22b-sfGFP-Y151TAG(氨苄霉素抗性)三个质粒共转化到大肠杆菌E.coli BL21(DE3)菌株中,经三抗性平板(卡那霉素,壮观霉素和氨苄霉素抗性)筛选出同时转化了三种质粒的阳性菌株,命名为表达菌株BL21-pET22b-sfGFP-Y151TAG。
实施例8.定点***非天然氨基酸的sfGFP的表达表达纯化和质谱验证
将实施例7中的BL21-pET22b-sfGFP-Y151TAG挑单菌落与5mL含有卡那霉素,壮观霉素和氨苄霉素的2YT培养基中过夜培养,次日按照1:100体积比例转接到新鲜的50mL含有卡那霉素,壮观霉素和氨苄霉素的2YT培养基中,待培养到指数生长初期,在培养基中添加1mM的前体小分子以及0.5mM的IPTG,诱导表达12-16小时后收集菌体。
将上述收集的表达菌体用高盐缓冲buffer(20mM Tris-HCl,300mM氯化钠pH8.0)重悬,在0-4℃超声破碎1-2min。将破碎产物进行高速离心(12000rpm,30分钟4℃),吸取上清。将上清样品与Ni亲和柱在4℃孵育30分钟,然后分别用25mM咪唑冲洗5-10个柱体积,40mM咪唑冲洗3个柱体积,250mM咪唑洗脱并浓缩,浓缩后通过SDS-PAGE蛋白电泳确定纯化蛋白质量。
将纯化的含有非天然氨基酸的绿色荧光蛋白用质谱(Electrospray IonizationMass Spectroscopy)验证非天然氨基酸的定点***,进而通过蛋白质谱确定非天然氨基酸的合成。检测的芳香巯/硒基前体分子顺序为化合物7、31、37、25、38、21、5、35、10、22、3、4、52、36。
如图8A至图8N所示依次为上述芳香巯/硒基前体分子生物合成非天然氨基酸的***sfGFP效率的质谱表征,质谱结果表明非天然氨基酸确实***到了sfGFP蛋白中。采用本发明提供的方法制备的大肠杆菌工程菌株确实可以表达带有非天然氨基酸的重组蛋白。
实施例9制备含Herceptin-Fab的质粒
1.从药物库(DRUGBANK)搜索Herceptin-Fab的重链和轻链的蛋白质序列,并添加胞间质转移的StII信号肽分别在重链和轻链的前面并由公司合成带有StII信号肽的Herceptin-Fab质粒(Herceptin-Fab基因3’端带有用于纯化蛋白的6xHis tag序列),然后将Herceptin-Fab的重链和轻链采用Gibson组装连接克隆至提前通过PCR线性化的pET22b载体,得到pET22b-Herceptin-Fab质粒。
StII信号肽可将在胞内表达出来的蛋白转移到胞间质通过二硫键组装,由于胞内属于还原环境,二硫键不会稳定存在,而胞间质可以稳定存在二硫键,进而可纯化得到正确折叠组装的fab。
pET22b-Herceptin-Fab质粒构建的具体步骤为:利用Fab-F,Fab-R引物从带有StII信号肽的Herceptin-Fab质粒中扩增Herceptin-Fab基因片段,将pET22b载体通过PCR方式线性化。
其中,Fab-F引物的序列如Seq ID No.36所示:GGGTATCTAGAGGTTGAGGTGATTTTATGAAAAAGAATATCGCATTTCTTCTTGCTAG;Fab-R引物的序列如Seq ID No.37所示:GCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCT,划线部分为6xHis标签;带有StII信号肽的Herceptin-Fab质粒的序列如Seq ID No.38所示。PCR反应体系如表10所示。
表10 PCR反应体系
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
Fab-F(10μM) | 2μL |
Fab-R(10μM) | 2μL |
带有StII信号肽的Herceptin-Fab的质粒(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃20s,30个循环;4℃保存。PCR产物经电泳,纯化,回收得到带有StII信号肽的Herceptin-Fab片段。
pET22b质粒进行PCR线性化的具体过程为:利用pET22-Fab-F和pET22-Fab-R引物,以pET22b载体为模板进行PCR扩增。其中,pET22-Fab-F引物序列如Seq ID No.39所示:GATCCGGCTGCTAACAAAGC;pET22-Fab-R引物序列如Seq ID No.40所示:AAAATCACCTCAACCTCTAGATACCC。PCR反应体系如表11所示。
表11 pET22b载体进行PCR线性化的PCR反应体系:
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
pET22-Fab-F(10μM) | 2μL |
pET22-Fab-R(10μM) | 2μL |
pET22b质粒(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃60s,30个循环;4℃保存PCR产物经电泳,纯化,回收得到线性化的pET22b质粒。
Gibson反应体系为:Herceptin-Fab基因片段(20ng/μL):1.5μL,线性化的pET22b(50ng/μL):1μL,Gibson反应液:2.5μL,总体系为5μL。
Gibson反应条件为:50℃1小时。
Gibson反应液转化大肠杆菌DH10B,涂布于50μg/ml氨苄青霉素的LB平板,次日挑单菌落于5mL 2YT液体培养基,培养基中添加浓度为50μg/ml的氨苄青霉素,置于37℃,220转速摇床培养12小时,提质粒(按照试剂盒说明书操作),质粒经限制性酶切验证和测序确认为pET22b-Herceptin-Fab质粒。pET22b-Herceptin-Fab质粒图谱如图9所示,序列如SeqID No.41所示。
2.位点选择参照文献(Proc Natl Acad Sci,U S A.2012Oct 2;109(40):16101-6.)对Herceptin-Fab蛋白选择了重链恒定区的121位点,轻链恒定区的169位点或202位点,可分别作为定点突变的靶氨基酸。
通过QuikChange定点突变方法分别将A121位点、K169位点或S202位点密码子突变为终止密码子-TAG。具体过程如下:
Herceptin-Fab的A121位,K169位及S202位设计能够使编码所述氨基酸的相应密码子突变为TAG的引物,具体引物如下表12所示。
表12:突变引物列表
以pET22b-Herceptin-Fab质粒为模板,分别使用表12中121位点、169位点、202位点的引物进行PCR扩增,PCR扩增反应体系如表13、表14及表15所示所示。
表13 121位点的PCR反应体系
PCR反应条件为:98℃10s,54℃5s,68℃80s,15个循环;4℃保存。
表14 169位点的PCR反应体系
PCR反应条件为:98℃10s,54℃5s,68℃80s,15个循环;4℃保存。
表15 202位点的PCR反应体系
ddH2O | 20μL |
KOD OneTM PCR Master Mix | 25μL |
Fab-202-F(10μM) | 2μL |
Fab-202-R(10μM) | 2μL |
pET22b-Herceptin-Fab质粒(50ng/μL) | 1μL |
总体积 | 50μL |
PCR反应条件为:98℃10s,54℃5s,68℃80s,15个循环;4℃保存。
分别将上面121位点、169位点、202位点的PCR产物进行DpnI消化,DpnI内切酶酶切反应体系为:50μL PCR反应液,1μL DpnI内切酶;反应条件为:37℃2小时。
消化后的片段分别直接转化DH5α菌株:具体转化步骤为:将5μL DpnI酶切产物加入大肠杆菌DH10B化转感受态中,冰浴30min,42℃热激45秒,冰浴3分钟,加入2YT培养基37℃孵育1小时后,分别涂在氨苄霉素抗性的平板上,次日挑取单克隆测序,以得到突变质粒pET22b-Herceptin-Fab-A121TAG,pET22b-Herceptin-Fab-K169TAG及pET22b-Herceptin-Fab-S202TAG的质粒。上述突变质粒分别与pET22b-Herceptin-Fab质粒仅有121位点、169位点、202位点密码子分别突变为TAG的差别。
实施例10.定点突变的Herceptin-Fab表达菌株的制备
将实施例2制备的pULTRA-PhSeRS质粒(壮观霉素抗性)和实施例8所制备的具有氨苄霉素抗性的表达质粒pET22-Herceptin-Fab-A121TAG或pET22-Herceptin-Fab-K169TAG或pET22-Herceptin-Fab-S202TAG同时转化到实施例3制备的菌株BL-pBK-CysM-NtSat4(卡那霉素抗性)中,经三抗性平板(卡那霉素,壮观霉素和氨苄霉素抗性)筛选出同时转化了三种表达质粒的阳性菌株,分别命名为表达菌株BL21-pET22-Herceptin-Fab-A121TAG,BL21-pET22-Herceptin-Fab-K169TAG,BL21-pET22-Herceptin-Fab-S202TAG。
同样的,采用上述方法将未突变的质粒pET22-Herceptin-Fab与PBK-CysM-NtSat4质粒和pULTRA-PhSeRS质粒共转化后得到未突变的野生型BL21-pET22-Herceptin-Fab表达菌株。
实施例11.Herceptin-Fab蛋白定点***生物合成的非天然氨基酸的重组蛋白的表达和纯化
1.定点***生物合成的非天然氨基酸的重组蛋白的表达
将实施例8中制备的BL21-pET22-Herceptin-Fab-A121TAG表达菌株在2YT培养基(含有50μg/ml壮观霉素,100μg/ml氨苄霉素和50μg/ml卡那霉素)中37℃过夜培养。次日按照1:50接种到新鲜2YT中至OD值为0.3时,加入1mM实施例5制备的对巯基叠氮(化合物22)继续培养,待OD值为0.6-1.0时,加入0.5mM IPTG,于25℃诱导表达16-18小时后收集菌体。此表达实验采用的阳性对照为在实施例8中构建的野生型的pET22-Herceptin-Fab表达菌株,除接种菌不同外,其余培养条件均与突变菌相同。BL21-pET22-Herceptin-Fab-K169TAG、BL21-pET22-Herceptin-Fab-S202TAG均按照以上培养条件进行表达。
2.定点***生物合成的非天然氨基酸的重组蛋白的表达
1)将上述收集的BL21-pET22-Herceptin-Fab-A121TAG菌体用高盐缓冲液(20mMTris-HCl,300mM氯化钠pH8.0)重悬,加入溶菌酶(1mg/ml)室温孵育2h,利用超声破碎1-2min。
2)将步骤1)破碎产物进行高速离心(12000rpm,30分钟4℃),吸取上清。
3)将步骤2)的上清样品与Ni亲和柱在4℃孵育30分钟,然后分别用25mM咪唑冲洗5-10个柱体积,40mM咪唑冲洗3个柱体积,250mM咪唑洗脱并浓缩,浓缩后进行SDS-PAGE蛋白电泳确定纯化蛋白质量。
将纯化的含有非天然氨基酸的Herceptin-Fab蛋白用质谱(ElectrosprayIonization Mass Spectroscopy)验证非天然氨基酸的定点***,进而通过蛋白质谱确定非天然氨基酸的合成,结果如图10所示。从图10可以看出,该Herceptin-Fab蛋白中确实***了含对巯基叠氮的非天然氨基酸。
实施例12.定点***生物合成的非天然氨基酸的Herceptin-Fab蛋白的偶联反应
如图11所示为定点***生物合成的非天然氨基酸的Herceptin-Fab蛋白可以与荧光物质、药物、PEG进行偶联的结构图。如图12所示为实施例11制备的定点***生物合成的非天然氨基酸的Herceptin-Fab蛋白的偶联原理示意图,为了简便表示,图12中将聚乙二醇(PEG)偶联和荧光偶联放在一起,实际偶联仅仅为一种偶联剂与突变蛋白作用。
1.聚乙二醇(PEG)偶联
将实施例11制备的含叠氮官能团的A121TAG突变的Herceptin-Fab蛋白(与DBCO-mPEG20K(购买自西安瑞禧生物科技有限公司)按摩尔比1:300混合,在pH为7.4的PBS缓冲液中室温反应过夜。
PEG通过与DBCO连接,而DBCO与突变蛋白上的叠氮基进行反应,得到PEG修饰的Herceptin-Fab蛋白,将偶联PEG的蛋白进行SDS-PAGE电泳结果如图13所示。Herceptin-Fab蛋白经过PEG修饰后可以提高此抗体的半衰期,促进靶向HER2阳性癌细胞的治疗或诊疗成像的效果。
2.荧光偶联
将含叠氮官能团的A121TAG突变的Herceptin-Fab蛋白与DBCO-Cy5(购买自西安瑞禧生物科技有限公司)按摩尔比1:3(Herceptin-Fab:DBCO-Cy5)混合,在pH为7.4的PBS缓冲液中室温反应过夜。用偶联荧光基团Cy5的Fab来标记HER2阳性细胞SK BR-3,共聚焦显微镜观察***叠氮基团的Herceptin抗体片段对her2高表达细胞SK-BR-3的结合,结果如图14所示(左图:利用核染料Hoechst33342对细胞核进行染色,进而实现镜下的细胞定位;中图:通过Herceptin Fab上的叠氮基团,与DBCO-cy5反应后得到了荧光标记蛋白,将该荧光蛋白与细胞共孵育,镜下观察到细胞膜表面高表达的her2受体被染色;右图:Hoechst33342与cy5染色图叠加,显示细胞核与细胞膜的共定位)。从图14可以看出,将Herceptin-Fab蛋白与荧光基团偶联确实可用于肿瘤细胞成像。
采用上述方法可以将其他位点的突变体蛋白与DBCO修饰的PEG,药物或者荧光偶联,得到近似产率的偶联产物。
除DBCO基团外,非天然氨基酸定点突变蛋白还可以与羟胺衍生物、DBCO衍生物、降冰片烯衍生物、BCN衍生物、TCO衍生物、环丙烯基团或反式环辛烯基团反应。
所述非天然氨基酸定点突变蛋白的位点为不影响结构稳定、活性并且为非天然氨基酸***效率较高的位点,可以根据现有研究报道进行选择。如前面所制备的突变后的sfGPF产量能够达到45-70mg/L,胞间质表达的突变后的抗体Fab能够得到1.5mg/L的产量。这都与相应突变前野生型蛋白的产量相当,说明本方法通过生物合成非天然氨基酸并定点***蛋白质中,生产的蛋白效率与野生蛋白的生产效率相当,具有工业应用潜力。
采用本发明所提供的含芳基硫酚或芳基硒酚经修饰的氨基酸定点***的重组蛋白,通过含芳基硫酚或芳基硒酚经修饰的氨基酸的特定基团与荧光、药物、PEG等实现偶联,当重组蛋白为抗体时,该抗体和荧光偶联可以实现肿瘤成像;该抗体和药物偶联时,可以实现对肿瘤的定点杀伤;该抗体或者一些治疗蛋白和PEG修饰剂偶联时,可增加抗体或者治疗蛋白的半衰期,可提高药物的效率。
虽然本发明所揭露的实施方式如上,但所述的内容仅为便于理解本发明而采用的实施方式,并非用以限定本发明。任何本发明所属领域内的技术人员,在不脱离本发明所揭露的精神和范围的前提下,可以在实施的形式及细节上进行任何的修改与变化,但本发明的专利保护范围,仍须以所附的权利要求书所界定的范围为准。
SEQUENCE LISTING
<110> 北京大学
<120> 含芳基硫酚或芳基硒酚经修饰的氨基酸、重组蛋白及其生物合成方法及应用
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gcgcagtcac caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact 420
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agcgcacgag ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 720
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cggataacgg cagtgaagtg tggttaaaac tggaaggcaa taacccggca ggttcggtga 240
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cgctgaaagg ctatcgcatg aaattgctga tgcccgacaa catgagccag gaacgccgtg 420
cggcgatgcg tgcttatggt gcggaactga ttcttgtcac caaagagcag ggcatggaag 480
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tcaataatcc cgataaccct tatgcgcatt acaccaccac tgggccggaa atctggcagc 600
aaaccggcgg gcgcatcact cattttgtct ccagcatggg gacgaccggc actatcaccg 660
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aagagggcag cagcattccc ggcattcgcc gctggcctac ggaatatctg ccggggattt 780
tcaacgcttc tctggtggat gaggtgctgg atattcatca gcgcgatgcg gaaaacacca 840
tgcgcgaact ggcggtgcgg gaaggaatat tctgtggcgt cagctccggc ggcgcggttg 900
ccggagcact gcgggtggca aaagctaacc ctgacgcggt ggtggtggcg atcatctgcg 960
atcgtggcga tcgctacctt tctaccgggg tgtttgggga agagcatttt agccaggggg 1020
cggggattga ctacaaggac gacgatgaca agtgactgca gtttcaaacg ctaaattgcc 1080
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tgtaggccgg ataaggcgtt cacgccgcat ccggcaagaa acagcaaaca atccaaaacg 1200
ccgcgttcag cggcgttttt tctgcttttc ttcgcgaatt aattccgctt cgcacatgtg 1260
agcaaaaggc cagcaaaagg ccaggaaccg tttgagatcg ttttggtctg cgcgtaatct 1320
cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta 1380
ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt 1440
cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg 1500
gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg 1560
gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga 1620
actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg 1680
aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg 1740
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat 1800
ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaag gatctcaaga agatcctttg 1860
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 1920
atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat 1980
tcaacgggaa acgtcttgct cgaggccgcg attaaattcc aacatggatg ctgatttata 2040
tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta 2100
tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga 2160
tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat 2220
caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa 2280
aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct 2340
ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga 2400
tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag 2460
tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa 2520
gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct 2580
tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga 2640
ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca 2700
gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca 2760
tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag 2820
cattacgctg acttgacggg acggcggctt tgttgaataa atcgaggatc 2870
<210> 9
<211> 978
<212> DNA
<213> 人工序列
<400> 9
atggctgcgg cgaccccgcc gaccaacccg ctgagccgtg acccgaacaa gccgcagatc 60
gataaccacg tgtacaacta cgttaagttt tgccgtccga gctttccgga actggttagc 120
tgcgcgccga tcccggagaa gaacagcaaa attggtcgta acgaggaaga ggacgatctg 180
tggctgaaga tgaaagacga agcgcgtagc gacatcgatc aagagccgat tctgagcacc 240
tactatatca ccagcattct ggcgcacgat agcatggaac gtgcgctggc gaaccacctg 300
agcatgaagc tgagcaacag cagcctgccg agcagcaccc tgtacgacct gtttctgggc 360
gtgctgaccg aagattgcag ccaggatatc attaaggcgg tgatcgcgga cctgcgtgcg 420
gttaaagagc gtgatccggc gtgcattagc tatgttcact gcttcctgaa ctttaagggt 480
ttcctggcgt gccaagcgca ccgtatcgcg cacaaactgt ggagcaacgg ccgtcagatc 540
ctggcgctgc tgattcaaaa ccgtgttagc gaagtgttcg cggttgacat tcacccgggt 600
gcgaagatcg gtaaaggcat tctgctggat catgcgaccg gtgtggttgt gggtgaaacc 660
gcggtgatcg gcaacaacgt tagcattctg cacaacgtga ccctgggtgg caccggcaag 720
atcagcggcg accgtcaccc gaaaatcggt gatggcgttc tgattggtgc gggcacctgc 780
gtgctgggta acgttatcat tgaggacggc gcgaaaatcg gtgcgggcag cgttgtgctg 840
aagaaagtgc cggcgcgtac caccgcggtt ggtaacccgg cgcgtctgct gggtggcaag 900
gaaaacccga agaaactgga taaaatcccg agcctgacca tggaccacac ctacgagtgg 960
agcgattatg ttatttaa 978
<210> 10
<211> 3688
<212> DNA
<213> 人工序列
<400> 10
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatggctgcg gcgaccccgc cgaccaaccc gctgagccgt gacccgaaca 480
agccgcagat cgataaccac gtgtacaact acgttaagtt ttgccgtccg agctttccgg 540
aactggttag ctgcgcgccg atcccggaga agaacagcaa aattggtcgt aacgaggaag 600
aggacgatct gtggctgaag atgaaagacg aagcgcgtag cgacatcgat caagagccga 660
ttctgagcac ctactatatc accagcattc tggcgcacga tagcatggaa cgtgcgctgg 720
cgaaccacct gagcatgaag ctgagcaaca gcagcctgcc gagcagcacc ctgtacgacc 780
tgtttctggg cgtgctgacc gaagattgca gccaggatat cattaaggcg gtgatcgcgg 840
acctgcgtgc ggttaaagag cgtgatccgg cgtgcattag ctatgttcac tgcttcctga 900
actttaaggg tttcctggcg tgccaagcgc accgtatcgc gcacaaactg tggagcaacg 960
gccgtcagat cctggcgctg ctgattcaaa accgtgttag cgaagtgttc gcggttgaca 1020
ttcacccggg tgcgaagatc ggtaaaggca ttctgctgga tcatgcgacc ggtgtggttg 1080
tgggtgaaac cgcggtgatc ggcaacaacg ttagcattct gcacaacgtg accctgggtg 1140
gcaccggcaa gatcagcggc gaccgtcacc cgaaaatcgg tgatggcgtt ctgattggtg 1200
cgggcacctg cgtgctgggt aacgttatca ttgaggacgg cgcgaaaatc ggtgcgggca 1260
gcgttgtgct gaagaaagtg ccggcgcgta ccaccgcggt tggtaacccg gcgcgtctgc 1320
tgggtggcaa ggaaaacccg aagaaactgg ataaaatccc gagcctgacc atggaccaca 1380
cctacgagtg gagcgattat gttatttaaa tcggatcccg ggcccgtcga ctgcagaggc 1440
ctgcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 1500
ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 1560
tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 1620
ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 1680
gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 1740
gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 1800
ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 1860
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 1920
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 1980
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 2040
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 2100
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 2160
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2220
gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2280
tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 2340
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 2400
agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 2460
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 2520
attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 2580
agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 2640
atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 2700
cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 2760
ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 2820
agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 2880
tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 2940
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 3000
caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 3060
ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 3120
gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 3180
tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 3240
tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 3300
cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 3360
cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 3420
gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 3480
atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 3540
agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 3600
ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 3660
aataggcgta tcacgaggcc ctttcgtc 3688
<210> 11
<211> 71
<212> DNA
<213> 人工序列
<400> 11
aaaaaaatat tctcaacata aaaaactttg tgtaatactt gtactcaagg aggatggctg 60
cggcgacccc g 71
<210> 12
<211> 68
<212> DNA
<213> 人工序列
<400> 12
taaatcgaac ttttgctgag ttgaaggatc aaaaaaatat tctcaacata aaaaactttg 60
tgtaatac 68
<210> 13
<211> 81
<212> DNA
<213> 人工序列
<400> 13
aagcgggaca ggctgacaac ccgaggatcc aaaacgccgc tgaacgcggc gttttttaaa 60
taacataatc gctccactcg t 81
<210> 14
<211> 19
<212> DNA
<213> 人工序列
<400> 14
ggatcctcgg gttgtcagc 19
<210> 15
<211> 25
<212> DNA
<213> 人工序列
<400> 15
gatccttcaa ctcagcaaaa gttcg 25
<210> 16
<211> 3981
<212> DNA
<213> 人工序列
<400> 16
ctcgggttgt cagcctgtcc cgcttataag attgttgaca attaatcatc cggctcgtat 60
aatgtgtgga attgtcagcg gttaacaatt tcacacagga aacagaccaa ggagcccata 120
tgagtacatt agaacaaaca ataggcaata cgcctctggt gaagttgcag cgaatggggc 180
cggataacgg cagtgaagtg tggttaaaac tggaaggcaa taacccggca ggttcggtga 240
aagatcgtgc ggcactttcg atgatcgtcg aggcggaaaa gcgcggggaa attaaaccgg 300
gtgatgtctt aatcgaagcc accagtggta acaccggcat tgcgctggca atgattgccg 360
cgctgaaagg ctatcgcatg aaattgctga tgcccgacaa catgagccag gaacgccgtg 420
cggcgatgcg tgcttatggt gcggaactga ttcttgtcac caaagagcag ggcatggaag 480
gtgcgcgcga tctggcgctg gagatggcga atcgtggcga aggaaagctg ctcgatcagt 540
tcaataatcc cgataaccct tatgcgcatt acaccaccac tgggccggaa atctggcagc 600
aaaccggcgg gcgcatcact cattttgtct ccagcatggg gacgaccggc actatcaccg 660
gcgtctcacg ctttatgcgc gaacaatcca aaccggtgac cattgtcggc ctgcaaccgg 720
aagagggcag cagcattccc ggcattcgcc gctggcctac ggaatatctg ccggggattt 780
tcaacgcttc tctggtggat gaggtgctgg atattcatca gcgcgatgcg gaaaacacca 840
tgcgcgaact ggcggtgcgg gaaggaatat tctgtggcgt cagctccggc ggcgcggttg 900
ccggagcact gcgggtggca aaagctaacc ctgacgcggt ggtggtggcg atcatctgcg 960
atcgtggcga tcgctacctt tctaccgggg tgtttgggga agagcatttt agccaggggg 1020
cggggattga ctacaaggac gacgatgaca agtgactgca gtttcaaacg ctaaattgcc 1080
tgatgcgcta cgcttatcag gcctacatga tctctgcaat atattgagtt tgcgtgcttt 1140
tgtaggccgg ataaggcgtt cacgccgcat ccggcaagaa acagcaaaca atccaaaacg 1200
ccgcgttcag cggcgttttt tctgcttttc ttcgcgaatt aattccgctt cgcacatgtg 1260
agcaaaaggc cagcaaaagg ccaggaaccg tttgagatcg ttttggtctg cgcgtaatct 1320
cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta 1380
ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt 1440
cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg 1500
gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg 1560
gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga 1620
actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg 1680
aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg 1740
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat 1800
ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaag gatctcaaga agatcctttg 1860
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 1920
atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat 1980
tcaacgggaa acgtcttgct cgaggccgcg attaaattcc aacatggatg ctgatttata 2040
tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta 2100
tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga 2160
tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat 2220
caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa 2280
aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct 2340
ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga 2400
tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag 2460
tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa 2520
gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct 2580
tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga 2640
ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca 2700
gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca 2760
tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag 2820
cattacgctg acttgacggg acggcggctt tgttgaataa atcgaacttt tgctgagttg 2880
aaggatcaaa aaaatattct caacataaaa aactttgtgt aatacttgta ctcaaggagg 2940
atggcggcgt gcattgacac ctgccgtacc ggtaaaccgc agatcagccc gcgtgatagc 3000
agcaagcacc acgacgatga gagcggtttt cgttacatga actatttccg ttacccggac 3060
cgtagcagct ttaacggcac ccaaaccaaa accctgcaca cccgtccgct gctggaggac 3120
ctggatcgtg acgcggaagt ggacgatgtt tgggcgaaga ttcgtgagga agcgaaaagc 3180
gatatcgcga aggagccgat tgtgagcgcg tactatcacg cgagcatcgt tagccaacgt 3240
agcctggaag cggcgctggc gaacaccctg agcgtgaaac tgagcaacct gaacctgccg 3300
agcaacaccc tgttcgacct gtttagcggt gttctgcaag gcaacccgga tattgtggag 3360
agcgttaaac tggacctgct ggcggtgaag gaacgtgatc cggcgtgcat cagctatgtt 3420
cactgcttcc tgcactttaa aggtttcctg gcgtgccagg cgcaccgtat tgcgcacgag 3480
ctgtggaccc aggatcgtaa gatcctggcg ctgctgattc aaaaccgtgt gagcgaagcg 3540
tttgcggttg acttccaccc gggcgcgaag atcggtaccg gcattctgct ggatcacgcg 3600
accgcgatcg tgattggcga gaccgcggtg gttggcaaca acgtgagcat cctgcacaac 3660
gttaccctgg gtggcaccgg taaacagtgc ggcgatcgtc acccgaagat cggtgacggc 3720
gttctgattg gtgcgggcac ctgcatcctg ggtaacatca ccattggtga aggcgcgaaa 3780
attggtgcgg gcagcgtggt tctgaaagat gtgccgccgc gtaccaccgc ggttggtaac 3840
ccggcgcgtc tgctgggtgg caaggataac ccgaaaaccc acgacaagat cccgggcctg 3900
accatggacc aaaccagcca cattagcgaa tggagcgatt acgtgatcta aaaaacgccg 3960
cgttcagcgg cgttttggat c 3981
<210> 17
<211> 1011
<212> DNA
<213> 人工序列
<400> 17
atggcggcgt gcattgacac ctgccgtacc ggtaaaccgc agatcagccc gcgtgatagc 60
agcaagcacc acgacgatga gagcggtttt cgttacatga actatttccg ttacccggac 120
cgtagcagct ttaacggcac ccaaaccaaa accctgcaca cccgtccgct gctggaggac 180
ctggatcgtg acgcggaagt ggacgatgtt tgggcgaaga ttcgtgagga agcgaaaagc 240
gatatcgcga aggagccgat tgtgagcgcg tactatcacg cgagcatcgt tagccaacgt 300
agcctggaag cggcgctggc gaacaccctg agcgtgaaac tgagcaacct gaacctgccg 360
agcaacaccc tgttcgacct gtttagcggt gttctgcaag gcaacccgga tattgtggag 420
agcgttaaac tggacctgct ggcggtgaag gaacgtgatc cggcgtgcat cagctatgtt 480
cactgcttcc tgcactttaa aggtttcctg gcgtgccagg cgcaccgtat tgcgcacgag 540
ctgtggaccc aggatcgtaa gatcctggcg ctgctgattc aaaaccgtgt gagcgaagcg 600
tttgcggttg acttccaccc gggcgcgaag atcggtaccg gcattctgct ggatcacgcg 660
accgcgatcg tgattggcga gaccgcggtg gttggcaaca acgtgagcat cctgcacaac 720
gttaccctgg gtggcaccgg taaacagtgc ggcgatcgtc acccgaagat cggtgacggc 780
gttctgattg gtgcgggcac ctgcatcctg ggtaacatca ccattggtga aggcgcgaaa 840
attggtgcgg gcagcgtggt tctgaaagat gtgccgccgc gtaccaccgc ggttggtaac 900
ccggcgcgtc tgctgggtgg caaggataac ccgaaaaccc acgacaagat cccgggcctg 960
accatggacc aaaccagcca cattagcgaa tggagcgatt acgtgatcta a 1011
<210> 18
<211> 73
<212> DNA
<213> 人工序列
<400> 18
tcaaaaaaat attctcaaca taaaaaactt tgtgtaatac ttgtactcaa ggaggatggc 60
ggcgtgcatt gac 73
<210> 19
<211> 68
<212> DNA
<213> 人工序列
<400> 19
taaatcgaac ttttgctgag ttgaaggatc aaaaaaatat tctcaacata aaaaactttg 60
tgtaatac 68
<210> 20
<211> 79
<212> DNA
<213> 人工序列
<400> 20
aagcgggaca ggctgacaac ccgaggatcc aaaacgccgc tgaacgcggc gttttttaga 60
tcacgtaatc gctccattc 79
<210> 21
<211> 3631
<212> DNA
<213> 人工序列
<400> 21
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatggacgaa tttgaaatga taaagagaaa cacatctgaa attatcagcg 480
aggaagagtt aagagaggtt ttaaaaaaag atgaaaaatc tgcttggata ggttttgaac 540
caagtggtaa aatacattta gggcattatc tccaaataaa aaagatgatt gatttacaaa 600
atgctggatt tgatataatt atacatttgg ctgatttagg agcctattta aaccagaaag 660
gagagttgga tgagattaga aaaataggag attataacaa aaaagttttt gaagcaatgg 720
ggttaaaggc aaaatatgtt tatggaagtg aaaatagtct tgataaggat tatacactga 780
atgtctatag attggcttta aaaactacct taaaaagagc aagaaggagt atggaactta 840
tagcaagaga ggatgaaaat ccaaaggttg ctgaagttat ctatccaata atgcaggtta 900
atagtattca ttatgagggc gttgatgttg cagttggagg gatggagcag agaaaaatac 960
acatgttagc aagggagctt ttaccaaaaa aggttgtttg tattcacaac cctgtcttaa 1020
cgggtttgga tggagaagga aagatgagtt cttcaaaagg gaattttata gctgttgatg 1080
actctccaga agagattagg gctaagataa agaaagcata ctgcccagct ggagttgttg 1140
aaggaaatcc aataatggag atagctaaat acttccttga atatccttta accataaaaa 1200
ggccagaaaa atttggtgga gatttgacag ttaatagcta tgaggagtta gagagtttat 1260
ttaaaaataa ggaattgcat ccaatgcggt tgaagaatgc tgtagctgaa gaacttataa 1320
agattttaga gccaattaga aagagattat aaatcggatc ccgggcccgt cgactgcaga 1380
ggcctgcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 1440
ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 1500
taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 1560
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 1620
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 1680
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 1740
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 1800
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 1860
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 1920
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 1980
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2040
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2100
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2160
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2220
aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 2280
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 2340
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 2400
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 2460
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 2520
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 2580
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 2640
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 2700
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 2760
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 2820
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 2880
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 2940
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 3000
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 3060
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 3120
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 3180
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 3240
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 3300
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 3360
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 3420
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 3480
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 3540
tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 3600
aaaaataggc gtatcacgag gccctttcgt c 3631
<210> 22
<211> 50
<212> DNA
<213> 人工序列
<400> 22
caaaggaggt gcggccgcat ggacgaattt gaaatgataa agagaaacac 50
<210> 23
<211> 60
<212> DNA
<213> 人工序列
<400> 23
accgtttaaa cgcggccgct tataatctct ttctaattgg ctctaaaatc tttataagtt 60
<210> 24
<211> 18
<212> DNA
<213> 人工序列
<400> 24
gcggccgcgt ttaaacgg 18
<210> 25
<211> 18
<212> DNA
<213> 人工序列
<400> 25
gcggccgcac ctcctttg 18
<210> 26
<211> 4977
<212> DNA
<213> 人工序列
<400> 26
accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc 60
cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttagggagct 120
gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac 180
aaaggaggtg cggccgcatg gacgaatttg aaatgataaa gagaaacaca tctgaaatta 240
tcagcgagga agagttaaga gaggttttaa aaaaagatga aaaatctgct tggataggtt 300
ttgaaccaag tggtaaaata catttagggc attatctcca aataaaaaag atgattgatt 360
tacaaaatgc tggatttgat ataattatac atttggctga tttaggagcc tatttaaacc 420
agaaaggaga gttggatgag attagaaaaa taggagatta taacaaaaaa gtttttgaag 480
caatggggtt aaaggcaaaa tatgtttatg gaagtgaaaa tagtcttgat aaggattata 540
cactgaatgt ctatagattg gctttaaaaa ctaccttaaa aagagcaaga aggagtatgg 600
aacttatagc aagagaggat gaaaatccaa aggttgctga agttatctat ccaataatgc 660
aggttaatag tattcattat gagggcgttg atgttgcagt tggagggatg gagcagagaa 720
aaatacacat gttagcaagg gagcttttac caaaaaaggt tgtttgtatt cacaaccctg 780
tcttaacggg tttggatgga gaaggaaaga tgagttcttc aaaagggaat tttatagctg 840
ttgatgactc tccagaagag attagggcta agataaagaa agcatactgc ccagctggag 900
ttgttgaagg aaatccaata atggagatag ctaaatactt ccttgaatat cctttaacca 960
taaaaaggcc agaaaaattt ggtggagatt tgacagttaa tagctatgag gagttagaga 1020
gtttatttaa aaataaggaa ttgcatccaa tgcggttgaa gaatgctgta gctgaagaac 1080
ttataaagat tttagagcca attagaaaga gattataagc ggccgcgttt aaacggtctc 1140
cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa 1200
cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct 1260
gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 1320
catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 1380
ggccttgttt gtgagctccc ggtcatcaat catccccata atccttgtta gcctgcaggt 1440
aattccgctt cgcaacatgt gagcaccggt ttattgacta ccggaagcag tgtgaccgtg 1500
tgcttctcaa atgcctgagg ccagtttgct caggctctcc ccgtggaggt aataattgac 1560
gatatgatca gtgcacggct aactaagcgg cctgctgact ttctcgccga tcaaaaggca 1620
ttttgctatt aagggattga cgagggcgta tctgcgcagt aagatgcgcc ccgcattccg 1680
gcggtagttc agcagggcag aacggcggac tctaaatccg catggcaggg gttcaaatcc 1740
cctccgccgg accaaattcg aaaagcctgc tcaacgagca ggcttttttg catgctcgag 1800
cagctcaggg tcgaatttgc catggcggcc accaggtacc accggcgcct caggcatttg 1860
agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat 1920
aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcatcgtgg ccggatcttg 1980
cggcccctcg gcttgaacga attgttagac attatttgcc gactaccttg gtgatctcgc 2040
ctttcacgta gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt 2100
cttgtccaag ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc 2160
gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt 2220
accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg 2280
agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat 2340
caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca 2400
gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat 2460
tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt 2520
cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag 2580
ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg 2640
tcaccgtaac cagcaaatca atatcactgt gtggcttcag gccgccatcc actgcggagc 2700
cgtacaaatg tacggccagc aacgtcggtt cgagatggcg ctcgatgacg ccaactacct 2760
ctgatagttg agtcgatact tcggcgatca ccgcttccct catactcttc ctttttcaat 2820
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2880
agaaaaataa acaaatagct agctcactcg gtcgctacgc tccgggcgtg agactgcggc 2940
gggcgctgcg gacacataca aagttaccca cagattccgt ggataagcag gggactaaca 3000
tgtgaggcaa aacagcaggg ccgcgccggt ggcgtttttc cataggctcc gccctcctgc 3060
cagagttcac ataaacagac gcttttccgg tgcatctgtg ggagccgtga ggctcaacca 3120
tgaatctgac agtacgggcg aaacccgaca ggacttaaag atccccaccg tttccggcgg 3180
gtcgctccct cttgcgctct cctgttccga ccctgccgtt taccggatac ctgttccgcc 3240
tttctccctt acgggaagtg tggcgctttc tcatagctca cacactggta tctcggctcg 3300
gtgtaggtcg ttcgctccaa gctgggctgt aagcaagaac tccccgttca gcccgactgc 3360
tgcgccttat ccggtaactg ttcacttgag tccaacccgg aaaagcacgg taaaacgcca 3420
ctggcagcag ccattggtaa ctgggagttc gcagaggatt tgtttagcta aacacgcggt 3480
tgctcttgaa gtgtgcgcca aagtccggct acactggaag gacagatttg gttgctgtgc 3540
tctgcgaaag ccagttacca cggttaagca gttccccaac tgacttaacc ttcgatcaaa 3600
ccacctcccc aggtggtttt ttcgtttaca gggcaaaaga ttacgcgcag aaaaaaagga 3660
tctcaagaag atcctttgat cttttctact gaaccgctct agatttcagt gcaatttatc 3720
tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag 3780
tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc 3840
gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg 3900
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 3960
gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac 4020
agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt 4080
tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg 4140
tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg 4200
gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga 4260
acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg 4320
ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc 4380
agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga 4440
cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata 4500
ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca 4560
gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg 4620
cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc 4680
atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt 4740
tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg 4800
cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc 4860
actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc 4920
tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattc 4977
<210> 27
<211> 3451
<212> DNA
<213> 人工序列
<400> 27
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatgagcaaa ggagaagaac ttttcactgg agttgtccca attcttgttg 480
aattagatgg tgatgttaat gggcacaaat tttctgtccg tggagagggt gaaggtgatg 540
ctacaaacgg aaaactcacc cttaaattta tttgcactac tggaaaacta cctgttccgt 600
ggccaacact tgtcactact ctgacctatg gtgttcaatg cttttcccgt tatccggatc 660
acatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca 720
ctatatcttt caaagatgac gggacctaca agacgcgtgc tgaagtcaag tttgaaggtg 780
atacccttgt taatcgtatc gagttaaagg gtattgattt taaagaagat ggaaacattc 840
ttggacacaa actcgagtac aactttaact cacacaatgt atacatcacg gcagacaaac 900
aaaagaatgg aatcaaagct aacttcaaaa ttcgccacaa cgttgaagat ggttccgttc 960
aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag 1020
acaaccatta cctgtcgaca caatctgtcc tttcgaaaga tcccaacgaa aagcgtgacc 1080
acatggtcct tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagctct 1140
acaaaggatc ccaccaccac caccaccact aaatcggatc ccgggcccgt cgactgcaga 1200
ggcctgcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 1260
ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 1320
taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 1380
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 1440
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 1500
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 1560
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 1620
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 1680
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 1740
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 1800
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 1860
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 1920
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 1980
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2040
aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 2100
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 2160
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 2220
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 2280
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 2340
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 2400
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 2460
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 2520
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 2580
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 2640
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 2700
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 2760
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 2820
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 2880
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 2940
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 3000
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 3060
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 3120
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 3180
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 3240
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 3300
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 3360
tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 3420
aaaaataggc gtatcacgag gccctttcgt c 3451
<210> 28
<211> 5608
<212> DNA
<213> 人工序列
<400> 28
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160
cctctagagt ttgacagcat tatcatcgat ctcgagaaat cataaaaaat ttatttgctt 5220
tgtgagcgga taacaattat aatagattca attgtgagcg gataacaatt tcacacagaa 5280
ttcattaaag aggagaaatt acataagctt aattagctga gcttggactc ctgttgatag 5340
atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg 5400
cgttttttat tggtgagaat ccaagctagc ttggcggcgg ccgcactcga gcaccaccac 5460
caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc 5520
accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 5580
ttgctgaaag gaggaactat atccggat 5608
<210> 29
<211> 60
<212> DNA
<213> 人工序列
<400> 29
cacacagaat tcattaaaga ggagaaatta catatgagca aaggagaaga acttttcact 60
<210> 30
<211> 50
<212> DNA
<213> 人工序列
<400> 30
gtccaagctc agctaattaa gcttttagtg gtggtggtgg tggtgggatc 50
<210> 31
<211> 24
<212> DNA
<213> 人工序列
<400> 31
aagcttaatt agctgagctt ggac 24
<210> 32
<211> 33
<212> DNA
<213> 人工序列
<400> 32
atgtaatttc tcctctttaa tgaattctgt gtg 33
<210> 33
<211> 6349
<212> DNA
<213> 人工序列
<400> 33
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160
cctctagagt ttgacagcat tatcatcgat ctcgagaaat cataaaaaat ttatttgctt 5220
tgtgagcgga taacaattat aatagattca attgtgagcg gataacaatt tcacacagaa 5280
ttcattaaag aggagaaatt acatatgagc aaaggagaag aacttttcac tggagttgtc 5340
ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt ccgtggagag 5400
ggtgaaggtg atgctacaaa cggaaaactc acccttaaat ttatttgcac tactggaaaa 5460
ctacctgttc cgtggccaac acttgtcact actctgacct atggtgttca atgcttttcc 5520
cgttatccgg atcacatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat 5580
gtacaggaac gcactatatc tttcaaagat gacgggacct acaagacgcg tgctgaagtc 5640
aagtttgaag gtgataccct tgttaatcgt atcgagttaa agggtattga ttttaaagaa 5700
gatggaaaca ttcttggaca caaactcgag tacaacttta actcacacaa tgtatacatc 5760
acggcagaca aacaaaagaa tggaatcaaa gctaacttca aaattcgcca caacgttgaa 5820
gatggttccg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct 5880
gtccttttac cagacaacca ttacctgtcg acacaatctg tcctttcgaa agatcccaac 5940
gaaaagcgtg accacatggt ccttcttgag tttgtaactg ctgctgggat tacacatggc 6000
atggatgagc tctacaaagg atcccaccac caccaccacc actaaaagct taattagctg 6060
agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca 6120
gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagctag cttggcggcg 6180
gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag 6240
gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 6300
aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat 6349
<210> 34
<211> 46
<212> DNA
<213> 人工序列
<400> 34
tttaactcac acaatgtata gatcacggca gacaaacaaa agaatg 46
<210> 35
<211> 49
<212> DNA
<213> 人工序列
<400> 35
gtttgtctgc cgtgatctat acattgtgtg agttaaagtt gtactcgag 49
<210> 36
<211> 58
<212> DNA
<213> 人工序列
<400> 36
gggtatctag aggttgaggt gattttatga aaaagaatat cgcatttctt cttgctag 58
<210> 37
<211> 43
<212> DNA
<213> 人工序列
<400> 37
gctttgttag cagccggatc tcagtggtgg tggtggtggt gct 43
<210> 38
<211> 4245
<212> DNA
<213> 人工序列
<400> 38
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatgaaaaag aatatcgcat ttcttcttgc tagcatgttc gttttttcta 480
ttgctacaaa cgcatacgct gacatccaga tgacccagtc tccatcctcc ctgtctgcat 540
ctgtaggaga cagagtcacc atcacttgcc gggcaagtca ggatgtgaat accgcggtcg 600
catggtatca gcagaaacca gggaaagccc ctaagctcct gatctattct gcatccttct 660
tgtatagtgg ggtcccatca aggttcagtg gcagtagatc tgggacagat ttcactctca 720
ccatcagcag tctgcaacct gaagattttg caacttacta ctgtcaacag cattacacta 780
cccctccgac gttcggccaa ggtaccaagc ttgagatcaa acgaactgtg gctgcaccat 840
ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgtcgtgt 900
gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg gataacgccc 960
tccaatcggg taactcccag gagagtgtca cagagcagga cagcaaggac agcacctaca 1020
gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa gtctacgcct 1080
gcgaagtcac ccatcagggc ctgtcctcgc ccgtcacaaa gagcttcaac aggggagagt 1140
gttaagctgg ggatcctcta gaggttgagg tgattttatg aaaaagaata tcgcatttct 1200
tcttgcatct atgttcgttt tttctattgc tacaaacgcg tacgctgagg tgcagctggt 1260
ggagtctgga ggaggcttgg tccagcctgg ggggtccctg agactctcct gtgcagcctc 1320
tgggttcaat attaaggaca cttacatcca ctgggtccgc caggctccag ggaaggggct 1380
ggagtgggtc gcacgtattt atcctaccaa tggttacaca cgctacgcag actccgtgaa 1440
gggccgattc accatctccg cagacacttc caagaacacg gcgtatcttc aaatgaacag 1500
cctgagagcc gaggacacgg ccgtgtatta ctgttcgaga tggggcggtg acggcttcta 1560
tgccatggac tactggggcc aaggaaccct ggtcaccgtc tcctcagcct ccaccaaggg 1620
cccatcggtc ttccccctgg caccctcctc caagagcacc tctgggggca cagcggccct 1680
gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc 1740
cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct 1800
cagcagcgtg gtgactgtgc cctctagcag cttgggcacc cagacctaca tctgcaacgt 1860
gaatcacaag cccagcaaca ccaaggtgga caagaaagtt gagcccaaat cttgtgacaa 1920
aactcacaca gcggccgcac tcgagcacca ccaccaccac cactgaatcg gatcccgggc 1980
ccgtcgactg cagaggcctg catgcaagct tggcgtaatc atggtcatag ctgtttcctg 2040
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 2100
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 2160
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 2220
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 2280
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 2340
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 2400
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 2460
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 2520
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 2580
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 2640
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 2700
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 2760
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 2820
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 2880
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 2940
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 3000
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 3060
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 3120
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 3180
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 3240
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 3300
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 3360
taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 3420
tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3480
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 3540
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 3600
aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 3660
cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 3720
tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 3780
gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 3840
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 3900
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 3960
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 4020
cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 4080
agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4140
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 4200
tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc 4245
<210> 39
<211> 20
<212> DNA
<213> 人工序列
<400> 39
gatccggctg ctaacaaagc 20
<210> 40
<211> 26
<212> DNA
<213> 人工序列
<400> 40
aaaatcacct caacctctag ataccc 26
<210> 41
<211> 6927
<212> DNA
<213> 人工序列
<400> 41
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160
cctctagaaa taattttgtt taactttaag aaggagaata catcaactag tacgcaagtt 5220
cacgtaaaaa gggtatctag aggttgaggt gattttatga aaaagaatat cgcatttctt 5280
cttgctagca tgttcgtttt ttctattgct acaaacgcat acgctgacat ccagatgacc 5340
cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 5400
agtcaggatg tgaataccgc ggtcgcatgg tatcagcaga aaccagggaa agcccctaag 5460
ctcctgatct attctgcatc cttcttgtat agtggggtcc catcaaggtt cagtggcagt 5520
agatctggga cagatttcac tctcaccatc agcagtctgc aacctgaaga ttttgcaact 5580
tactactgtc aacagcatta cactacccct ccgacgttcg gccaaggtac caagcttgag 5640
atcaaacgaa ctgtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg 5700
aaatctggaa ctgcctctgt cgtgtgcctg ctgaataact tctatcccag agaggccaaa 5760
gtacagtgga aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag 5820
caggacagca aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac 5880
tacgagaaac acaaagtcta cgcctgcgaa gtcacccatc agggcctgtc ctcgcccgtc 5940
acaaagagct tcaacagggg agagtgttaa gctggggatc ctctagaggt tgaggtgatt 6000
ttatgaaaaa gaatatcgca tttcttcttg catctatgtt cgttttttct attgctacaa 6060
acgcgtacgc tgaggtgcag ctggtggagt ctggaggagg cttggtccag cctggggggt 6120
ccctgagact ctcctgtgca gcctctgggt tcaatattaa ggacacttac atccactggg 6180
tccgccaggc tccagggaag gggctggagt gggtcgcacg tatttatcct accaatggtt 6240
acacacgcta cgcagactcc gtgaagggcc gattcaccat ctccgcagac acttccaaga 6300
acacggcgta tcttcaaatg aacagcctga gagccgagga cacggccgtg tattactgtt 6360
cgagatgggg cggtgacggc ttctatgcca tggactactg gggccaagga accctggtca 6420
ccgtctcctc agcctccacc aagggcccat cggtcttccc cctggcaccc tcctccaaga 6480
gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc cccgaaccgg 6540
tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc 6600
tacagtcctc aggactctac tccctcagca gcgtggtgac tgtgccctct agcagcttgg 6660
gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag gtggacaaga 6720
aagttgagcc caaatcttgt gacaaaactc acacagcggc cgcactcgag caccaccacc 6780
accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca 6840
ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt 6900
tgctgaaagg aggaactata tccggat 6927
<210> 42
<211> 43
<212> DNA
<213> 人工序列
<400> 42
ccctggtcac cgtctcctca tagtccacca agggcccatc ggt 43
<210> 43
<211> 43
<212> DNA
<213> 人工序列
<400> 43
accgatgggc ccttggtgga ctatgaggag acggtgacca ggg 43
<210> 44
<211> 44
<212> DNA
<213> 人工序列
<400> 44
gtgtcacaga gcaggacagc taggacagca cctacagcct cagc 44
<210> 45
<211> 44
<212> DNA
<213> 人工序列
<400> 45
gctgaggctg taggtgctgt cctagctgtc ctgctctgtg acac 44
<210> 46
<211> 44
<212> DNA
<213> 人工序列
<400> 46
gcgaagtcac ccatcagggc ctgtcctcgc ccgtcacaaa gagc 44
<210> 47
<211> 44
<212> DNA
<213> 人工序列
<400> 47
gctctttgtg acgggcgagg acaggccctg atgggtgact tcgc 44
Claims (12)
1.一种重组蛋白,其特征在于,所述重组蛋白包含含芳基硫酚或芳基硒酚经修饰的氨基酸的一个或多个残基,该含芳基硫酚或芳基硒酚经修饰的氨基酸具有如下式(1)所示通式:
式(1);
或其盐或其前药,其中,A为S或者Se,m和n相同或不同,m和n为0-3之间的整数;U为(C0-C4)直链或者支链烷基;
G为叠氮基,乙酰基,四嗪基,2-氧代丙酰胺基团,硼酸基,醛基,氰基,三氟甲基或巯基;
含G官能团的键仅在苯环的邻位,间位或者对位;G1,G2和G3中的一个可以是-N-,或者-C-,且G1,G2和G3中的其余者独立的为-CH-;
其中,当G分别取自叠氮基,醛基,氰基时,U为C0烷基;
当G分别取自硼酸基,巯基时,m为0,同时U为C0烷基。
2.如权利要求1所述的重组蛋白,其特征在于,A为S。
3.如权利要求1所述的重组蛋白,其特征在于,A为Se。
4.如权利要求1所述的重组蛋白,其特征在于,G为叠氮基,具有如下式(2)所示通式:
式(2);
其中,m为0-3之间的整数;
叠氮基的键仅在苯环的邻位,间位或者对位,或者同时存在于任意两个相同或不同位置。
5.如权利要求1所述的重组蛋白,其特征在于,G为乙酰基,具有如下式(3)所示通式:
式(3);
其中,m为0-3之间的整数;
乙酰基的键仅在苯环的邻位,间位或者对位,或者同时存在于任意两个相同或不同位置。
6.如权利要求1所述的重组蛋白,其特征在于,G为四嗪基,具有如下式(4)所示通式:
式(4);
其中, m为0-3之间的整数;
四嗪基的键仅在苯环的邻位,间位或者对位,或者同时存在于任意两个相同或不同位置。
7.如权利要求1所述的重组蛋白,其特征在于,所述含芳基硫酚或芳基硒酚经修饰的氨基酸具体为如下式S-1至S-70或Se-1至Se-69中的任一式的化合物:
。
8.一种偶联物,其特征在于,包含连接于偶联剂的如权利要求1-7任一项所述的重组蛋白,且所述偶联物包含所述重组蛋白和所述偶联剂之间的连接部分;
其中,所述偶联剂为荧光分子或PEG,连接部分为羟胺衍生物或DBCO衍生物。
9.如权利要求1-7任一项所述的重组蛋白的制备方法,其特征在于,包括如下步骤:
1)构建带有半胱氨酸合成酶CysM基因和O-乙酰丝氨酸转移酶NtSat4基因或AtSat3基因的辅助质粒
a. 使用带有持续性表达启动子Trc1序列的半胱氨酸合成酶CysM基因的引物从大肠杆菌基因组中扩增得到半胱氨酸合成酶CysM基因,使得该半胱氨酸合成酶CysM基因的前面融合Trc1启动子;将该半胱氨酸合成酶CysM基因通过Gibson组装方法克隆到含有p15a复制起点的大肠杆菌pBK表达载体中,该载体含有卡那霉素抗性基因,得到pBK-CysM质粒;
b. 合成带有O-乙酰丝氨酸转移酶NtSat4基因的质粒或带有O-乙酰丝氨酸转移酶AtSat3基因的质粒,使用带有持续性表达启动子IPP序列的NtSat4基因的引物或带有持续性表达启动子IPP序列的AtSat3基因的引物从带有NtSat4或AtSat3基因的质粒中扩增得到带有持续性表达启动子IPP的NtSat4或AtSat3基因的片段;将该NtSat4基因或AtSat3基因的片段通过Gibson组装方式克隆到pBK-CysM载体中,得到辅助质粒pBK-CysM-NtSat4或pBK-CysM-AtSat3;
2)制备表达含芳基硫酚或芳基硒酚经修饰的氨基酸的菌株
将质粒pBK-CysM-NtSat4或质粒pBK-CysM-AtSat3转化到BL21(DE3)中,得到合成含芳基硫酚或芳基硒酚经修饰的氨基酸的菌株BL-pBK-CysM-NtSat4或BL-pBK-CysM-AtSat3;
3)含芳基硫酚或芳基硒酚经修饰的氨基酸的合成
将菌株BL-pBK-CysM-NtSat4或BL-pBK-CysM-AtSat3接种在5 mL含有50 µg/ml卡那霉素的2YT培养基中,过夜培养,次日按照接种量为1:100体积比转接于20 mL含有50 µg/ml卡那霉素的2YT培养基中,培养至对数生长初期,添加1 mM芳香巯/硒基前体分子,在37℃,220转速的摇床中培养12小时;吸取1 mL菌液,12000转速离心2分钟,吸取培养基上清,用0.22µm滤膜过滤,分离纯化氨基酸;
其中,步骤1)所述的带有持续性表达启动子Trc1序列的半胱氨酸合成酶CysM基因的引物由上游引物CysM-Trc1-F1、CysM-Trc1-F2、CysM-Trc1-F3和下游引物CysM-R组成;
其中,CysM-Trc1-F1的核苷酸序列如Seq ID No.1所示;CysM-Trc1-F2的核苷酸序列如Seq ID No.2所示;CysM-Trc1-F3的核苷酸序列如Seq ID No.3所示;CysM-R的核苷酸序列如Seq ID No.4所示;
含有p15a复制起点的大肠杆菌pBK表达载体的核苷酸序列如Seq ID No.5;
带有持续性表达启动子IPP序列的NtSat4基因的引物由上游引物NtSat4-F1、NtSat4-F2和下游引物NtSat4-R组成;
其中,NtSat4-F1的核苷酸序列如Seq ID No.11所示;NtSat4-F2的核苷酸序列如SeqID No.12所示;NtSat4-R的核苷酸序列如Seq ID No.13所示;
带有持续性表达启动子IPP序列的AtSat3基因的引物由上游引物AtSat3-F1、AtSat3-F2和下游引物AtSat3-R组成;
其中,AtSat3-F1的核苷酸序列如Seq ID No.18所示;AtSat3-F2的核苷酸序列如SeqID No.19所示;AtSat3-R的核苷酸序列如Seq ID No.20所示;
带有O-乙酰丝氨酸转移酶NtSat4基因的质粒的核苷酸序列如Seq ID No.10所示;NtSat4基因的核苷酸序列如Seq ID No.9所示;AtSat3基因的核苷酸序列如Seq ID No.17所示;
4)构建含有可***含芳基硫酚或芳基硒酚经修饰的氨基酸的包含氨酰tRNA合成酶基因和tRNA基因的辅助质粒pUltra-PhSeRS/tRNA
合成包含突变位点Y32W,L65H,H70G,F108N,Q109S,D158S和L162E的氨酰tRNA合成酶PhSeRS基因的质粒,将PhSeRS基因替换pUltra-CNF质粒中的CNF片段,其中pUltra-CNF质粒已包含tRNA基因序列,得到带有可***含芳基硫酚或芳基硒酚经修饰的氨基酸的辅助质粒pUltra-PhSeRS/tRNA;
5)构建含有终止密码子突变的表达目标蛋白的质粒
将C端带有6xHis标签的目标蛋白的基因序列用Gibson组装方法克隆到pET22b载体中,利用Quikchange定点突变的方法将特定位点的密码子突变为终止密码子TAG,得到带有TAG的目标蛋白的质粒;
6)表达具有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的大肠杆菌工程菌株
将步骤1)制备的辅助质粒pBK-CysM-NtSat4或pBK-CysM-AtSat3、步骤4)制备的辅助质粒pUltra-PhSeRS/tRNA、步骤5)制备的带有TAG的目标蛋白的质粒共转化到大肠杆菌BL21(DE3)中,得到表达具有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的大肠杆菌工程菌株;
或,将步骤4)制备的辅助质粒pUltra-PhSeRS/tRNA、步骤5)制备的带有TAG的目标蛋白的质粒共转化到步骤2)制备的菌株BL-pBK-CysM-NtSat4或BL-pBK-CysM-AtSat3中,得到表达具有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白的大肠杆菌工程菌株;
7)将所述大肠杆菌工程菌株培养至对数生长初期,添加1 mM芳香巯/硒基前体分子,使用终浓度为0.5 mM 的IPTG诱导表达12-20小时,得到含有定点***含芳基硫酚或芳基硒酚经修饰的氨基酸的重组蛋白,然后用Ni-NTA Agarose进行纯化;
所述芳香巯/硒基前体分子为具有如式(5)的化学结构的通式化合物;
式(5);
其中,A为SH或者SeH,m和n相同或不同,m和n为0-3之间的整数;U为(C0-C4)直链或者支链烷基;
G取自叠氮基,乙酰基,四嗪基,2-氧代丙酰胺基团,硼酸基,醛基,氰基,三氟甲基,巯基,硝基,羧甲基,胺乙酰基,氨基,甲氧基,羟基或卤素;
含G官能团的键可以仅在苯环的邻位,间位或者对位;G1,G2和G3中的一个可以是-N-,或者-C-,且G1,G2和G3中的其余者独立的是-CH-;
其中,当G分别取自叠氮基,醛基,氰基时,U为C0烷基;
当G分别取自硼酸基,巯基时,m为0,同时U为C0烷基;
其中,所述卤素为-F、-Cl或-Br。
10.如权利要求9所述的重组蛋白的制备方法,其特征在于,所述包含突变位点Y32W,L65H,H70G,F108N,Q109S,D158S和L162E的PhSeRS基因的质粒的核苷酸序列如Seq IDNo.21所示。
11.如权利要求9所述的重组蛋白的制备方法,其特征在于,所述大肠杆菌工程菌株培养的培养条件为:含有50 µg/ml壮观霉素,100 µg/ml氨苄霉素和50 µg/ml 卡那霉素的2-YT培养基在37℃条件下220 rpm进行培养;对数生长初期为OD=0.3。
12.如权利要求8所述的偶联物在作为荧光成像剂中的应用。
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