CN114716563A - 一种融合蛋白及其制备与应用 - Google Patents
一种融合蛋白及其制备与应用 Download PDFInfo
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- CN114716563A CN114716563A CN202110010261.1A CN202110010261A CN114716563A CN 114716563 A CN114716563 A CN 114716563A CN 202110010261 A CN202110010261 A CN 202110010261A CN 114716563 A CN114716563 A CN 114716563A
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Abstract
本发明提供了一种融合蛋白,其从N端到C端依次包括:SEC2突变体、连接短肽和iNGR,所述SEC2突变体在如SEQ ID NO:18所示序列的第20、22、118和/或122位氨基酸残基发生突变,所述连接短肽的氨基酸序列如SEQ ID NO:4所示,所述iNGR的氨基酸序列如SEQ ID NO:5所示。本发明还提供了该融合蛋白的制备方法和应用。本发明的靶向性融合蛋白可特异性靶向并浸润到肿瘤组织微环境,大幅度提升了超抗原的肿瘤特异性和血管通透性,提高了对肿瘤的杀伤效率,从而用于治疗患者时可有效提高患者的存活能力,具有良好的临床应用价值。
Description
技术领域
本发明涉及基因工程领域,具体涉及一种融合蛋白及其制备与应用。
背景技术
肿瘤的免疫治疗,是指应用免疫学的原理和方法,通过激活体内的免疫细胞和增强机体抗肿瘤免疫应答,从而有效杀伤肿瘤细胞,抑制肿瘤细胞生长的治疗方法。由于其副作用小、治疗效果明显,正逐渐成为未来肿瘤治疗的发展方向。
靶向性融合蛋白是在现代医学生物学发展的基础上提出的一种新型的治疗策略,由靶向分子和细胞毒性分子结合而成。靶向分子常选用肿瘤特异性抗体或短肽,将能够对目的细胞产生杀伤的效应分子递送至肿瘤患处,符合目前肿瘤精准治疗发展趋势。
iNGR是一种具有环状结构的靶向性穿膜肽,分子量较小,且具有较高的水溶性,序列为CRNGRGPDC,其中的NGR序列能够特异性地结合肿瘤组织血管内皮及肿瘤细胞高表达的整合素αv,然后被特定蛋白酶切割之后,残基片段具有R/KXXR/K的结构特点,能够与NRP-1(神经纤毛蛋白-1)相互作用,介导发生细胞膜穿透效应。iNGR因其功能多样而广泛应用于抗肿瘤药物、肿瘤成像剂以及一些生物制品等的靶向载体的研究。其中,整合素αv为细胞黏附受体家族成员,调节多种细胞功能,尤其是在实体瘤的发生、发展和转移,其表达也与肿瘤的恶性程度正相关。NRP-1作为神经系统的调节因子,在肿瘤局部调节VEGF诱导血管生成中扮演重要的角色。其与肿瘤细胞的生长、迁移及血管生成等密切相关。
目前大部分研究都采用iNGR和化学药物进行联用,但化学药物需要用脂质体包载,之后才能与iNGR进行偶联,这类联合药物取得了不错的抑瘤效果。但是,由于脂质体包载的化学药物需要在组织内进行释放才能发挥药物作用,所以脂质体的类型、方式对药物效果有极大的影响。较低的脂质体包封率一直是困扰脂质体药物的关键,且脂质体类药物还存在稳定性较差,易被机体主动清除等问题。这样也导致了iNGR偶联的脂质体也存在结构及效果的不稳定等缺点,为其应用添加了阻碍。此外,在iNGR与化学药物联用的应用中,还有一些采用将iNGR与化学药物作为两个独立的部分混合给药,导致iNGR无法对化学药物发挥主动的靶向递送作用,仅能辅助部分聚集在肿瘤血管组织中的化药穿透进入肿瘤组织中。这不仅减弱了iNGR介导的效应分子在肿瘤局部的聚集效率,并且由于弱的靶向效果,导致化学药物对正常组织的毒副作用没有得到改善。
现有技术中还公开了将具有高渗透性的靶向性穿膜肽iNGR与其他TRAIL、CDD等蛋白类效应分子结合组成融合蛋白,取得的效果略佳。但是肿瘤脉管系统和肿瘤组织错综复杂,因此有待进一步寻求靶向性更强和组织穿透力更强的融合蛋白。
发明内容
本发明所要解决的技术问题是为了克服现有技术中融合蛋白不足以穿过肿瘤脉管系统和肿瘤组织复杂的微环境、导致抗肿瘤效果不够理想的技术缺陷,提供一种融合蛋白及其基因、其制备方法与应用。本发明将特定的靶向性穿膜肽与特定的超抗原通过特定的连接短肽进行连接,形成的融合蛋白可特异性靶向并浸润到高表达整合素αv与NRP-1的肿瘤组织微环境,大幅度提升了超抗原的肿瘤特异性和血管通透性,提高了对肿瘤的杀伤效率,从而用于治疗患者时可有效提高患者的存活能力,具有良好的临床应用价值。
本发明人对靶向分子、连接短肽、细胞毒性分子等进行了大量的研究,意外发现,当将靶向性穿膜肽iNGR与肿瘤免疫治疗药物超抗原中的SEC2突变体通过与连接短肽融合,配合特定的刚性的连接短肽(在构建融合蛋白中,一个关键的问题是两蛋白间的连接短肽Linker,即连接肽的长度对蛋白质的折叠和稳定性非常重要。如果接头序列太短,可能影响两蛋白高级结构的折叠,从而相互干扰;如果接头序列太长,又涉及免疫原性的问题,因为接头序列本身就是新的抗原)时,所得融合蛋白的肿瘤特异性和血管通透性显著增强。
为解决上述技术问题,本发明第一方面提供了一种融合蛋白,所述融合蛋白从N端到C端依次包括:SEC2突变体、连接短肽和iNGR,所述突变包括1~4个氨基酸的插入、缺失或替换,所述SEC2的氨基酸序列如SEQ ID NO:18所示,所述连接短肽的氨基酸序列如SEQ IDNO:4所示,所述iNGR的氨基酸序列如SEQ ID NO:5所示。
较佳地,所述SEC2突变体在如SEQ ID NO:18所示序列的第20、22、118和/或122位氨基酸残基发生突变(例如插入、缺失或替换)。
更佳地,所述SEC2突变体在如SEQ ID NO:18所示序列上具有T20L、G22E、H118A、H122A中的4、3、2或1个氨基酸取代。
更佳地,所述SEC2突变体的氨基酸序列如SEQ ID NO:3、SEQ ID NO:8或SEQ IDNO:11所示。
在某一较佳实施例中,所述SEC2突变体IAE-1即是指超级抗原蛋白金黄色葡萄球菌肠毒素C2(Staphylococcal enterotoxin C2,SEC2,氨基酸序列如SEQ ID NO:18所示)的第20位氨基酸残基T突变为L,第22位氨基酸残基G突变为E,第118位氨基酸残基H突变为A的SAg IAE-1异构体(本发明中均简称IAE-1,如SEQ ID NO:3所示)。
在某一较佳实施例中,所述SEC2突变体IAE-2即是指超级抗原蛋白金黄色葡萄球菌肠毒素C2(Staphylococcal enterotoxin C2,SEC2,氨基酸序列如SEQ ID NO:18所示)的第20位氨基酸残基T突变为L,第22位氨基酸残基G突变为E,第122位氨基酸残基H突变为A的SAg IAE-2异构体(本发明中均简称IAE-2,如SEQ ID NO:8所示)。
在某一较佳实施例中,所述SEC2突变体IAE-3即是指超级抗原蛋白金黄色葡萄球菌肠毒素C2(Staphylococcal enterotoxin C2,SEC2,氨基酸序列如SEQ ID NO:18所示)的第20位氨基酸残基T突变为L,第22位氨基酸残基G突变为E,第118位氨基酸残基H突变为A,第122位氨基酸残基H突变为A的SAg IAE-3异构体(本发明中均简称IAE-3,如SEQ ID NO:11所示)。
所述SEC2突变体也可参考专利申请CN201010275279.6,在此引用其全部内容并入本发明。
较佳地,所述融合蛋白的氨基酸序列如序列表中SEQ ID NO:2、SEQ ID NO:7、SEQID NO:10所示;更佳地,编码所述融合蛋白的核苷酸序列如序列表中SEQ ID NO:1、SEQ IDNO:6、SEQ ID NO:9所示。
为解决上述技术问题,本发明第二方面提供了一种融合基因,其编码如本发明第一方面所述的融合蛋白。
较佳地,其核苷酸序列如序列表中SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:9所示。
为解决上述技术问题,本发明第三方面提供了一种重组表达载体,所述重组表达载体中含如本发明第二方面所述的融合基因。
较佳地,所述重组表达载体的骨架载体为pET-28a-TEV。
为解决上述技术问题,本发明第四方面提供了一种转化体,其通过在宿主中导入如本发明第二方面所述的融合基因或者如本发明第三方面所述的重组表达载体即得所述转化体。
较佳地,所述宿主为大肠杆菌,优选为大肠杆菌E.coli BL21(DE3)细胞或者E.coli TG1。
为解决上述技术问题,本发明第五方面提供了一种融合蛋白的制备方法,其包括以下步骤:
(1)获得如本发明第四方面所述的转化体;
(2)筛选所述转化体,表达并纯化所述融合蛋白。
上述步骤(2)中,所述纯化优选包括将所述表达所得的菌体经超声波破碎后离心收集上清液,经过两次Ni亲合层析即可得到具有生物学活性的可溶性融合蛋白。通常情况下,两次Ni亲和层析后可以仅需收集上样的穿柱后的溶液即得融合蛋白。
较佳地,所述Ni亲合层析包括以0.2-0.8ml/min的上样速度将样品上样于预先平衡好的Ni亲合层析柱,使用8-12个柱体积的平衡缓冲液洗涤(洗去非特异性结合的杂蛋白即可)后用洗脱缓冲液洗脱即可,优选使用10个柱体积的平衡缓冲液洗涤;
更佳地,所述平衡缓冲液为含有20-80mM咪唑的平衡缓冲液,其组成优选为:20-30mM Tirs-HCl,800-1000mM NaCl,20-80mM咪唑,和/或,所述平衡缓冲液的pH值为7.2~8.0;和/或,所述洗脱缓冲液为含有250-300mM咪唑的洗脱缓冲液,其组成优选为:20-30mMTirs-HCl,800-1000mM NaCl,250-300mM咪唑;和/或,所述洗脱缓冲液的pH值为7.2~8.0。
较佳地,在所述的两次Ni亲合层析之间还包括超滤脱盐的步骤;更佳地,在所述超滤脱盐后还包括与TEV蛋白酶混合酶切的步骤;进一步更佳地,所述超滤脱盐后的产物与所述TEV蛋白酶的摩尔比为1:5;和/或,所述酶切的时间为24h。
为解决上述技术问题,本发明第六方面提供了一种如本发明第一方面所述的融合蛋白、如本发明第二方面所述的融合基因、如本发明第三方面所述的重组表达载体或如本发明第四方面所述的转化体在制备药物中的应用;优选在制备治疗肿瘤的药物中的应用,更优选在制备治疗肿瘤免疫的药物中的应用。
本发明某一方面还提供了一种如本发明第一方面所述的融合蛋白、如本发明第二方面所述的融合基因、如本发明第三方面所述的重组表达载体或如本发明第四方面所述的转化体在治疗肿瘤中的应用,更优选在治疗肿瘤免疫中的应用。
本发明中,“融合基因”指由两个或两个以上不同来源的核苷酸序列连接而成的基因,或者由同一来源但其天然位置并不互相连接的两个或两个以上核苷酸序列连接而成的基因。本发明的融合基因所编码的蛋白称作融合蛋白。
本发明中,所述的超抗原(superantigen,SAg)是一种在极低浓度下对T淋巴细胞产生极强免疫激活的蛋白质分子,其可在抗原递呈细胞外侧的抗原结合区与MHC II(组织相容性复合物)分子和T细胞Vβ区结合形成复合物,从而激活大量的T淋巴细胞增殖,使体外或体内释放大量的细胞因子和其它效应分子。
本申请所用氨基酸三字母代码和单字母代码如本领域技术人员知晓,或J.Biol.Chem,243,p3558(1968)中所述。
如本申请使用的,术语“包括”或“包含”旨在表示组合物和方法包括所述的元素但不排除其他元素,但根据上下文的理解,也包括“由……组成”的情况。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明通过将特定的靶向性穿膜肽与特定的超抗原突变体相连接形成的融合蛋白可特异性靶向并浸润到高表达整合素αv与NRP-1的肿瘤组织微环境,大幅度提升了超抗原的肿瘤特异性和血管通透性,增强其在肿瘤局部的富集,可显著抑制肿瘤细胞的生长,从而提升了抗肿瘤效果,应用到临床治疗时所需剂量随之可以下降,即用更低的剂量可以获得更好的抑瘤效果,而更低剂量会带来更低的毒性,从而降低药物使用过程中的副作用;此外,肿瘤特异性的提高可以使药物分子更多的集中在肿瘤区域,减少了其他非肿瘤区域和器官的分布量,从而也降低了毒性,提高患者的存活能力。在本发明某一较佳实施例中,本发明所述融合蛋白结合肿瘤细胞的能力较融合前的蛋白提高了5.09倍数以上,抑瘤率高达61.90%以上,将本发明的融合蛋白应用于患有黑色素瘤的小鼠时,小鼠的平均生存天数显著高于对照组。本发明融合蛋白的抑瘤效果显著优于“靶向性穿膜肽”和“超抗原”简单混用时的效果,即在抑瘤效果上取得了1+1>2的效果。
附图说明
图1为构建pET-28a-tev-iae-1-epapkp-iNGR酶切验证以1.0%琼脂糖凝胶电泳分析结果,其中:1为经过EcoR I和XhoI双酶切的pET-28a-tev-iae-1-epapkp-iNGR质粒;2为未经酶切的pET-28a-tev-iae-1-epapkp-iNGR质粒;M为λ-EcoT14 I/BglII digest DNAmarker;3和4分别为经过EcoRI和XhoI单酶切的pET-28a-tev-iae-1-epapkp-iNGR质粒。
图2为经AKTA Ni柱两次纯化后的可溶性表达的穿膜肽-超抗原融合蛋白IAE-1-EPAPKP-iNGR经12%的SDS-PAGE电泳分析图,其中:M为180KD蛋白Marker;1为经Ni柱层析纯化后透析除盐,再使用TEV蛋白酶酶切去除标签的二次纯化的融合蛋白,浓度为(300ng/μL);2为经Ni柱层析纯化后透析除盐的融合蛋白,浓度为(300ng/μL)。
图3为融合蛋白IAE-1-EPAPKP-NGR、IAE-1-EPAPKP-tLyp-1、IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR、IAE-1-GGGGS(G4S)-iNGR、IAE-1-(GS)5-iNGR、IAE-1-EPAPK-iNGR、iNGR-EPAPKP-IAE-1以及IAE-1体外结合αv+和NRP-1+小鼠黑色素瘤细胞B16F10的实验结果。
图4为融合蛋白IAE-1-EPAPKP-NGR、IAE-1-EPAPKP-tLyp-1、IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR、IAE-1-GGGGS(G4S)-iNGR、IAE-1-(GS)5-iNGR、IAE-1-EPAPK-iNGR、iNGR-EPAPKP-IAE-1以及IAE-1体外结合αv+和NRP-1+小鼠乳腺癌细胞4T1的实验结果。
图5为融合蛋白IAE-1-EPAPKP-NGR、IAE-1-EPAPKP-tLyp-1、IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR、iNGR-EPAPKP-IAE-1、IAE-1-GGGGS(G4S)-iNGR、IAE-1-(GS)5-iNGR、IAE-1-EPAPK-iNGR、sTRAIL-EPAPKP-iNGR以及IAE-1+iNGR、BSA、iNGR、IAE-1、IAE-2和IAE-3体外抑制αv+和NRP-1+小鼠黑色素瘤细胞微球B16F10的实验结果。
图6为融合蛋白IAE-1-EPAPKP-NGR、IAE-1-EPAPKP-tLyp-1、IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR、iNGR-EPAPKP-IAE-1、IAE-1-GGGGS(G4S)-iNGR、IAE-1-(GS)5-iNGR、IAE-1-EPAPK-iNGR、sTRAIL-EPAPKP-iNGR以及IAE-1+iNGR、BSA、iNGR、IAE-1、IAE-2和IAE-3体外抑制αv+和NRP-1+小鼠乳腺癌细胞微球4T1的实验结果。
图7为融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR以及生理盐水、IAE-1和IAE-1+iNGR体内抑制αv+和NRP-1+小鼠黑色素瘤细胞B16F10的实验结果。
图8为融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR以及生理盐水、IAE-1和IAE-1+iNGR体内抑制αv+和NRP-1+小鼠乳腺癌细胞4T1的实验结果。
图9为融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR、SEA-EPAPKP-iNGR、SEB-EPAPKP-iNGR、SEC2-EPAPKP-iNGR以及PBS和IAE-1体内抑制αv+和NRP-1+小鼠黑色素瘤细胞B16F10的生存曲线。
图10为融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR、SEA-EPAPKP-iNGR、SEB-EPAPKP-iNGR、SEC2-EPAPKP-iNGR以及PBS和IAE-1体内抑制αv+和NRP-1+小鼠乳腺癌细胞4T1的生存曲线。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
以下实施例中所有蛋白质分子的编码碱基序列DNA均由北京华大基因公司合成。
实施例1
1、融合蛋白IAE-1-EPAPKP-iNGR基因iae-1-epapkp-ingr,其具有如表1中的SEQID NO:1中的碱基序列,其中,IAE-1(IAE-1即是指超级抗原蛋白金黄色葡萄球菌肠毒素C2(Staphylococcal enterotoxin C2,SEC2,氨基酸序列为如SEQ ID NO:18所示的序列)的第20位氨基酸残基T突变为L,第22位氨基酸残基G突变为E,第118位氨基酸残基H突变为A的Sag IAE-1改构体,以下及说明书附图中均简称IAE-1)编码基因IAE-1具有如SEQ ID NO:1的第1位到717位所示的碱基序列,iNGR编码基因iNGR具有如SEQ ID NO:1的第736位到762位所示的碱基序列,通过编码连接短肽的DNA Linker EPAPKP具有如SEQ ID NO:1的第718位到735位所示的碱基序列。
表1
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
(1)SEQ ID NO:1的信息(参见序列表)
(a)序列特征:
长度:762bp
类型:核酸
链型:双链
拓扑结构:线性
(b)分子类型:cDNA
(c)假设:否
(d)反义:否
(2)融合基因IAE-1-epapkp-iNGR的制备:
(a)PCR引物设计及反应条件:根据pET28a载体(购自Novagen公司)多克隆位点结合上述sag异构体IAE-1设计正向引物(由北京华大基因公司合成),将epapkp-iNGR基因序列设计反向引物(由北京华大基因公司合成)进行PCR:
正向引物(F):5’-CGGAATTCGAGAGTCAACCAGACCC-3’(SEQ ID NO:33)
反向引物(R):
5’-CCCTCGAGTTAGCAATCCGGGCCGCGGCCGTTGCGGCAAGGTTTTGGCGCCGGTTCTCCATTCTTTGTTGTAAGGTGGACTTCTAT-3’(SEQ ID NO:34)
PCR反应体系为(Pyrobest buffer、dNTP、pyrobest DNA聚合酶均购自TAKARA公司):10×Pyrobest buffer 5μL、dNTP 250μmol、正反向引物各25pmol、模板为含sag基因的质粒DNA(序列如SEQ ID NO:1的第1位到711位的碱基序列所示)0.1μg、pyrobest DNA聚合酶2U,无菌超纯水补齐体积至50μL。
PCR反应条件为:第一阶段:95℃,5min;第二阶段:94℃,55s;60℃,2min;72℃,2min;共30个循环;第三阶段:72℃,10min。
(b)PCR产物回收:PCR扩增产物经1.0%琼脂糖凝胶电泳分析并切胶回收753bp的目的条带,操作方法按照江苏康为世纪有限公司胶回收纯化试剂盒使用说明进行。得到产物靶向性穿膜肽-超抗原融合基因IAE-1-epapkp-iNGR。
由SEQ ID NO:1所示的核苷酸所编码的融合蛋白IAE-1-EPAPKP-iNGR,具有如SEQID NO:2所示(具体见表1)的氨基酸序列。
其中,SEQ ID NO:2的信息如下
(a)序列特征
*长度:254残基
*类型:氨基酸
*链型:单链
*拓扑结构:线性
(b)分子类型:蛋白质
(c)假设:否
(d)反义:否
(e)最初来源:人工序列。
2、融合蛋白IAE-2-EPAPKP-iNGR基因IAE-2-epapkp-iNGR,其具有表2中的SEQ IDNO:6中的碱基序列,其中,IAE-2(IAE-2即是指SEC2的第20位氨基酸残基T突变为L,第22位氨基酸残基G突变为E,第122位氨基酸残基H突变为A的SAg IAE-2改构体,以下及说明书附图中均简称IAE-2)编码基因IAE-2具有SEQ ID NO:6的第1位到717位所示的碱基序列,iNGR编码基因iNGR具有如SEQ ID NO:6的第736位到762位所示的碱基序列,通过编码连接短肽的DNA LinkerEPAPKP具有如SEQ ID NO:6的第718位到735位所示的碱基序列。
表2
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
(1)SEQ ID NO:6的信息(参见表2)
(a)序列特征:
长度:762bp
类型:核酸
链型:双链
拓扑结构:线性
(b)分子类型:cDNA
(c)假设:否
(d)反义:否
(2)融合基因IAE-2-epapkp-iNGR的制备过程同上述第1部分(仅模板不一样)。由SEQ ID NO:6所示的核苷酸所编码的融合蛋白IAE-2-EPAPKP-iNGR,具有如SEQ ID NO:7所示(具体见表2)的氨基酸序列。其中,SEQ ID NO:7的信息如下:
(a)序列特征
*长度:254残基
*类型:氨基酸
*链型:单链
*拓扑结构:线性
(b)分子类型:蛋白质
(c)假设:否
(d)反义:否
(e)最初来源:人工序列。
3、融合蛋白IAE-3-EPAPKP-iNGR基因IAE-3-epapkp-iNGR,其具有表3中的SEQ IDNO:9中的碱基序列,其中,IAE-3(IAE-3即是指SEC2的第20位氨基酸残基T突变为L,第22位氨基酸残基G突变为E,第118位氨基酸残基H突变为A,第122位氨基酸残基H突变为A的SAgIAE-3改构体,以下及说明书附图中均简称IAE-3)编码基因IAE-3具有SEQ ID NO:9的第1位到717位所示的碱基序列,iNGR编码基因iNGR具有如SEQ ID NO:9的第736位到762位所示的碱基序列,通过编码连接短肽的DNA LinkerEPAPKP具有如SEQ ID NO:9的第718位到735位所示的碱基序列。
表3
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
(1)SEQ ID NO:9的信息(参见序列表)
(a)序列特征:
长度:762bp
类型:核酸
链型:双链
拓扑结构:线性
(b)分子类型:cDNA
(c)假设:否
(d)反义:否
(2)融合基因IAE-3-epapkp-iNGR的制备过程上述第1部分(仅模板不一样)。由SEQID NO:9所示的核苷酸所编码的融合蛋白IAE-3-EPAPKP-iNGR,具有如SEQ ID NO:10所示(具体见表3)的氨基酸序列。其中,SEQ ID NO:10的信息如下:
(a)序列特征
*长度:254残基
*类型:氨基酸
*链型:单链
*拓扑结构:线性
(b)分子类型:蛋白质
(c)假设:否
(d)反义:否
(e)最初来源:人工序列。
实施例2
将实施例1中所制得的三种靶向性穿膜肽-超抗原融合基因IAE-1-l-iNGR(即IAE-1-epapkp-iNGR)、IAE-2-l-iNGR(即IAE-2-epapkp-iNGR)、IAE-3-l-iNGR(即IAE-3-epapkp-iNGR)连接到原核表达载体pET-28a-TEV中,实现在大肠杆菌中表达靶向性穿膜肽-超抗原融合蛋白SAg-L-iNGR,具体为:
将融合基因sag-l-iNGR(即上述IAE-1-l-iNGR、IAE-2-l-iNGR、IAE-3-l-iNGR)连接入表达载体pET-28a-TEV(购自Novagen公司)中:将表达载体pET-28a-TEV的质粒DNA和sag-l-iNGR的基因DNA片段分别使用EcoRI(购自大连宝生物公司)和XhoI(购自大连宝生物公司)双酶切,经1.0%琼脂糖凝胶电泳,胶回收sag-l-iNGR片段和质粒pET-28a-TEV的DNA大片段,以T4DNA连接酶(购自大连宝生物公司)16℃连接过夜,构建靶向性穿膜肽-超抗原融合蛋白表达载体pET28a-TEV-sag-l-iNGR。连接产物转化大肠杆菌DH5α感受态细胞(购自大连宝生物公司)。以卡那青霉素(购自Sigma公司)抗性筛选转化子,挑选重组单克隆扩培,提取质粒DNA,经EcoR I和XhoI双酶切鉴定正确重组克隆(图1)。并将经双酶切验证正确的重组克隆质粒送往上海生工公司进行测序。将测序正确的质粒转化进入大肠杆菌BL21(DE3)感受态细胞中(购自北京天根生化科技公司)。
(1)融合蛋白SAg-L-iNGR(即IAE-1-L-iNGR(即IAE-1-EPAPKP-iNGR)、IAE-2-L-iNGR(即IAE-2-EPAPKP-iNGR)、IAE-3-L-iNGR(即IAE-3-EPAPKP-iNGR)的表达:接种上述转化重组质粒pET28a-TEV-sag-l-iNGR的BL21(DE3)单菌落于60μg/ml卡那青霉素的液体LB中37℃过夜,次日按1:100(体积比)转接到下一代,37℃培养至OD600为0.8,加入终浓度为10mMIPTG(购自Sigma公司)30℃和37℃诱导4h。
(2)收集含融合蛋白的上清:离心收集诱导表达后的菌体,每100ml原培养物的菌体重悬于10ml平衡缓冲液(20mMTirs-HCl,500mMNaCl,50mM咪唑,pH=7.9),于0℃超声破碎至菌液变清亮,100000rpm10min超高速离心(用10万的转数可以更好去除核酸、细胞碎片等杂质,对后续纯化处理有利,且实验发现10万转数并不会降低蛋白的收率)。收集上清。
(3)利用AKTA纯化仪(美国GE公司产品)纯化融合蛋白:离心后的上清液上样(上样速度为0.2-0.8ml/min)于AKTANi亲和层析柱(美国GE公司产品),使用十个柱体积的平衡缓冲液(20mMTirs-HCl,500mMNaCl,50mM咪唑,pH=7.9)漂洗,至UV检测数值稳定。最后使用洗脱缓冲液(20mMTirs-HCl,500mMNaCl,250mM咪唑,pH=7.9)洗脱目的蛋白,在UV检测数值开始拉升后进行收集,直至UV平稳。将收集的TEV-SAg-L-iNGR蛋白洗脱液进行透析除盐,并经过SDS-PAGE分析纯度,结果如图2所示的2号泳道所示。
(4)将透析后的融合蛋白TEV-SAg-L-iNGR与TEV蛋白酶(所使用的pET28a载体中是自带His-tag纯化标签的,故对融合蛋白TEV-SAg-L-iNGR进行亲和标签切割)以摩尔比1:5进行混合,酶切24h后,将混合体系上样AKTA纯化仪的Ni柱,收集上样时的第一次UV峰,即为融合蛋白SAg-L-iNGR,透析除盐并经过SDS-PAGE分析纯度,结果如图2所示的1号泳道所示,比2号泳道的纯度高。
实施例3靶向性穿膜肽-超抗原融合蛋白SAg-L-iNGR的肿瘤靶向性研究
选用western blot验证高表达整合素αv与NRP-1的鼠源性黑色素瘤细胞B16F10和鼠源性乳腺癌细胞4T1作为靶细胞(所用细胞购自“中国科学院典型培养物保藏委员会细胞库”)。
融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR的靶向性验证:使用荧光染料AlexaFlour 647(购自thermo公司)分别对融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR、和超抗原IAE-1进行了标记,将靶细胞B16F10及4T1固定后分别与上述荧光标记的蛋白进行混合孵育,孵育20min后,1000g离心5min,去上清。使用PBS重悬,离心。再次使用200μlPBS溶液重悬后,使用流式细胞仪进行检测。实验结果如图3和图4所示,图中显示荧光标记的IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR可以对靶细胞产生强的靶向结合作用,结合能力明显强于对照组,且三者之间的结合强度几乎无差异。
其中,IAE-1-EPAPKP-iNGR结合B16F10细胞的能力是IAE-1的5.58倍,IAE-2-EPAPKP-iNGR结合B16F10细胞的能力是IAE-1的5.09倍,IAE-3-EPAPKP-iNGR结合B16F10细胞的能力是IAE-1的5.51倍。
IAE-1-EPAPKP-iNGR结合4T1细胞的能力是IAE-1的9.21倍,IAE-2-EPAPKP-iNGR结合4T1细胞的能力是IAE-1的9.65倍,IAE-3-EPAPKP-iNGR结合4T1细胞的能力是IAE-1的9.49倍。
实施例4靶向性穿膜肽-超抗原融合蛋白SAg-L-iNGR的体外抗肿瘤活性研究
融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR的体外抗肿瘤活性验证:将B16F10和4T1细胞,以1×104cells/well加入U型细胞培养板(美国康宁4515型),培养48h形成稳定肿瘤微球后,按照1:10效靶比加入分离的单个小鼠脾细胞。再将实验组融合蛋白a.IAE-1-EPAPKP-iNGR、b.IAE-2-EPAPKP-iNGR、c.IAE-3-EPAPKP-iNGR、d.IAE-1、IAE-2、IAE-3分别单独使用、e.iNGR-EPAPKP-IAE-1(与a的区别为二者方向相反,从N端至C端依次为iNGR、EPAPKP和突变体IAE-1),f.iNGR,g.iNGR+IAE-1混合联用(即IAE-1+iNGR组),分别以相同的物质的量350pmol/μl加入各孔,同时设空白对照孔(仅加培养基RPMI-1640,美国Gibco公司产品)、肿瘤细胞对照孔(仅加肿瘤细胞),每样3个复孔。同样方法以牛血清白蛋白BSA(购自Sigma公司)为阴性对照设各孔。按常规条件(37℃、5%CO2浓度)培养48h后,每孔加入100μl Cell-tilter Glo 3D细胞活力检测试剂液(购自Promega公司)。室温放置25min后,使用酶标仪检测各孔内的生物发光。
抑瘤率(Tumor growth inhibition,%)=100-[(实验孔-空白对照孔)/(肿瘤细胞对照孔-空白对照孔)]×100。
实验结果显示(图5和图6):
在350pmol/μl浓度时,IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR对B16F10的抑瘤效果最为明显,分别达到了61.9%、66.55%、67.59%。其中IAE-1-EPAPKP-iNGR较IAE-1提高了28.73%、IAE-2-EPAPKP-iNGR较IAE-2提高了29.44%、IAE-3-EPAPKP-iNGR较IAE-3提高了31.68%,抑瘤率明显增强。
在350pmol/μl浓度时,IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR对4T1的抑瘤效果最为明显,分别达到了69.52%、67.47%、68.05%。其中IAE-1-EPAPKP-iNGR较IAE-1提高了38.32%、IAE-2-EPAPKP-iNGR较IAE-2提高了34.26%、IAE-3-EPAPKP-iNGR较IAE-3提高了36.15%,抑瘤率明显增强。
此外,更重要的是,针对两个细胞株的实验结果显示,IAE-1-EPAPKP-iNGR的抑瘤率不仅显著高于BSA对照组、iNGR单独使用组、IAE-1单独使用组,且显著高于iNGR+IAE-1联合治疗组,说明融合蛋白的抑瘤效果显著高于没有进行融合的两个分子混合在一起的联合治疗效果,说明蛋白的融合是非常关键的。
实施例5靶向性穿膜肽-超抗原融合蛋白SAg-L-iNGR的体内抗肿瘤实体瘤活性研究
融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR在小鼠体内抗肿瘤实体瘤活性验证:分别皮下接种106个/只黑色素瘤细胞B16F10于C57小鼠(北京维通利华实验动物技术有限公司,五周龄时进行接种)左侧背部,接种106个/只乳腺癌细胞4T1于BALB/c小鼠(北京维通利华实验动物技术有限公司,五周龄时进行接种)乳垫部位。待肿瘤长至约100mm3开始通过尾静脉给药,设置处理组a.IAE-1-EPAPKP-iNGR、b.IAE-2-EPAPKP-iNGR、c.IAE-3-EPAPKP-iNGR,d.IAE-1,e.iNGR与IAE-1混合联用(即IAE-1+iNGR组),使用生理盐水作为对照处理组,给药浓度为70pmol/只,每三天给药一次,共给药六次。记录给药期间肿瘤体积变化(图7和图8),并记录小鼠的死亡终点绘制生存曲线(图9和图10)。结果显示,融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR可以对高表达整合素αv与NRP-1的实体瘤产生特异性的靶向杀伤作用,其作用效果显著强于其他实验组(d.IAE-1,e.iNGR与IAE-1混合联用)及对照组。在给药终点day16时,各组的肿瘤抑制率如表4和表5所示:
表4.各个融合蛋白对B16F10造模的C57小鼠的肿瘤抑制率
*抑瘤率=(对照组肿瘤体积-实验组肿瘤体积)/对照组肿瘤体积*100%
表5.各融合蛋白对4T1造模的BALB/c小鼠的肿瘤抑制率
*抑瘤率=(对照组肿瘤体积-实验组肿瘤体积)/对照组肿瘤体积*100%
由表中可以看出:
IAE-1-EPAPKP-iNGR对B16F10造模的C57小鼠的肿瘤抑制率达到69.60%,IAE-2-EPAPKP-iNGR对B16F10造模的C57小鼠的肿瘤抑制率达到74.37%,IAE-3-EPAPKP-iNGR对B16F10造模的C57小鼠的肿瘤抑制率达到73.29%,显著性的高于IAE-1组的35.40%,也显著高于IAE-1+iNGR联用组的48.45%。
IAE-1-EPAPKP-iNGR对4T1造模的BALB/c小鼠的肿瘤抑制率达到75.45%,IAE-2-EPAPKP-iNGR对4T1造模的BALB/c小鼠的肿瘤抑制率达到73.36%,IAE-3-EPAPKP-iNGR对4T1造模的BALB/c小鼠的肿瘤抑制率达到80.85%,显著性的高于IAE-1组的42.85%,也显著高于IAE-1+iNGR联用组的56.55%。
给药小鼠的生存实验结果如图9和图10显示:
融合蛋白IAE-1-EPAPKP-iNGR给药组B16F10造模小鼠生存天数的中位数为29.5天,IAE-2-EPAPKP-iNGR给药组B16F10造模小鼠的平均生存天数为28.5天,IAE-3-EPAPKP-iNGR给药组B16F10造模小鼠的平均生存天数为29.5天,显著高于单独SAg蛋白(22.5天)与对照组(18.5天)。
融合蛋白IAE-1-EPAPKP-iNGR给药组4T1造模小鼠的平均生存天数为56.5天,IAE-2-EPAPKP-iNGR给药组4T1造模小鼠的平均生存天数为59.5天,IAE-3-EPAPKP-iNGR给药组4T1造模小鼠的平均生存天数为61天,显著高于单独SAg蛋白(44天)与对照组(34天)。
对比例:
(1)本发明合成并表达了以下几种融合蛋白:
本对比例中所有蛋白质分子的编码碱基序列DNA均由北京华大基因公司合成,所有蛋白分子的表达载体的构建、蛋白表达与纯化、蛋白定量方法,均与实施例2相同,所有蛋白分子的活性检测均与实施例3、4、5相同。
融合蛋白SEA-EPAPKP-iNGR具有下表6中的SEQ ID NO:13中的氨基酸序列,其编码基因sea-epapkp-iNGR,具有下表6中的SEQ ID NO:12中的碱基序列。
表6
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白SEB-EPAPKP-iNGR具有下表7中的SEQ ID NO:15中的氨基酸序列,其编码基因seb-epapkp-iNGR,具有下表7中的SEQ ID NO:14中的碱基序列。
表7
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白SEC2-EPAPKP-iNGR具有下表8中的SEQ ID NO:17中的氨基酸序列,其编码基因sec2-epapkp-iNGR,具有下表8中的SEQ ID NO:16中的碱基序列。
表8
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白IAE-1-GGGGS(G4S)-iNGR具有下表9中的SEQ ID NO:20中的氨基酸序列,其编码基因IAE-1-ggggs-iNGR,具有下表9中的SEQ ID NO:19中的碱基序列。
表9
注:加粗下划线的序列为超抗原改构体序列,粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白IAE-1-(GS)5-iNGR具有下表10中的SEQ ID NO:22中的氨基酸序列,其编码基因IAE-1-gsgsgsgsgs-iNGR,具有下表10中的SEQ ID NO:21中的碱基序列。
表10
注:加粗下划线的序列为超抗原改构体序列,粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白IAE-1-EPAPK-iNGR具有下表11中的SEQ ID NO:24中的氨基酸序列,其编码基因IAE-1-epapk-iNGR,具有下表11中的SEQ ID NO:23中的碱基序列。
表11
注:加粗下划线的序列为超抗原改构体序列,粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白iNGR-EPAPKP-IAE-1具有下表12中的SEQ ID NO:26中的氨基酸序列,其编码基因iNGR-epapkp-IAE-1,具有下表12中的SEQ ID NO:25中的碱基序列。
表12
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白IAE-1-EPAPKP-NGR具有下表13中的SEQ ID NO:28中的氨基酸序列,其编码基因IAE-1-epapkp-NGR,具有下表13中的SEQ ID NO:27中的碱基序列:
表13
注:粗体的序列为linker序列,下划线的序列为靶向分子序列NGR。
融合蛋白IAE-1-EPAPKP-tLyp-1具有下表14中的SEQ ID NO:30中的氨基酸序列,其编码基因IAE-1-epapkp-tlyp-1,具有下表14中的SEQ ID NO:29中的碱基序列:
表14
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
融合蛋白sTRAIL-EPAPKP-iNGR具有下表15中的SEQ ID NO:32中的氨基酸序列,其编码基因strail-epapkp-iNGR,具有下表15中的SEQ ID NO:31中的碱基序列:
表15
注:粗体的序列为linker序列,下划线的序列为靶向分子序列iNGR。
(2)为了对比对靶向相同受体的不同连接短肽的使用效果,本发明人构建了同靶点靶向分子NGR(针对靶点整合素αv)和tLyp-1(针对靶点神经纤毛蛋白NRP-1)连接超抗原后组成的融合蛋白a.IAE-1-EPAPKP-NGR,b.IAE-1-EPAPKP-tLyp-1,其中NGR具有靶向整合素αv的能力,tLyp-1具有靶向NRP-1并通过CendR来以增强药物肿瘤组织穿透性的能力。构建三种蛋白并纯化后,分别对他们的靶向验证实验和抗肿瘤活性进行了检验。
靶向验证实验方法同实施例3,结果如图3和图4所示,图中显示,在相同浓度时,IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR结合靶细胞B16F10和4T1的强度基本一致,而IAE-1-EPAPKP-iNGR结合B16F10细胞的能力是IAE-1-EPAPKP-NGR的2.13倍,是IAE-1-EPAPKP-tLyp-1的2.42倍。IAE-1-EPAPKP-iNGR结合4T1细胞的能力是IAE-1-EPAPKP-NGR的2.08倍,是IAE-1-EPAPKP-tLyp-1的2.37倍。说明了IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR三种融合蛋白对靶细胞有更强的结合力。
体外抗肿瘤活性验证实验方法同实施例4,结果如图5和图6显示,在350pmol/μl浓度时,IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR抑制靶细胞微球B16F10和4T1的生长的能力基本一致,而IAE-1-EPAPKP-iNGR对B16F10的抑瘤率较IAE-1-EPAPKP-NGR提高了22.8%,较IAE-1-EPAPKP-tLyp-1抑瘤率提高了18.61%。IAE-1-EPAPKP-iNGR对4T1的抑瘤率较IAE-1-EPAPKP-NGR提高了27.42%,较IAE-1-EPAPKP-tLyp-1抑瘤率提高了30.42%。这一体外抑瘤结果对比说明了可溶性融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR较其他两种靶向性短肽相比,能更好的携带效应分子渗透进入肿瘤微环境中发挥抑制作用。
(3)本发明对比了以不同方式连接组成的融合蛋白的靶向能力和体外抗肿瘤微球活性。为了对比iNGR连接短肽同SAg的不同连接方式,基于IAE-1-EPAPKP-iNGR(iNGR在Sag的C端)构建了将iNGR连接在超抗原Sag的N端的对比例融合蛋白iNGR-EPAPKP-IAE-1。为了对比本发明中的连接短肽(linker)与其他常用连接短肽的差异,构建了以G4S(一种柔性连接短肽,GGGGS)、(GS)5(一种柔性连接短肽)、EPAPK(比EPAPKP少一个氨基酸的刚性连接短肽)三种不同的Linker连接形成的融合蛋白IAE-1-GGGGS(G4S)-iNGR、IAE-1-(GS)5-iNGR、IAE-1-EPAPK-iNGR三种融合蛋白。以及将靶向分子iNGR和效应分子IAE-1作为两个独立个体的联用(IAE-1+iNGR)同样作为对比例。
靶向性验证的实验方法同实施例3,结果如图3和图4显示,在相同浓度时,IAE-1-EPAPKP-iNGR结合B16F10细胞的能力是IAE-1-GGGGS(G4S)-iNGR的1.66倍,是IAE-1-(GS)5-iNGR的1.68倍,是IAE-1-EPAPK-iNGR的2.43倍,是iNGR-EPAPKP-IAE-1的2.18倍。IAE-1-EPAPKP-iNGR结合4T1细胞的能力是IAE-1-GGGGS(G4S)-iNGR的1.84倍,是IAE-1-(GS)5-iNGR的1.60倍,是IAE-1-EPAPK-iNGR的1.79倍,是iNGR-EPAPKP-IAE-1的1.75倍。这些对比数据证明了:相对于使用其他linker而言,使用EPAPKP作为Linker时组成的融合蛋白能够更好的发挥靶细胞结合作用;此外,相对于将iNGR连接到超抗原的N端而言,将iNGR连接到超抗原的C端能使融合蛋白更好的发挥靶细胞结合作用。
体外抗肿瘤微球的实验方法同实施例4,结果如图5和图6显示,在350pmol/μl浓度时,IAE-1-EPAPKP-iNGR对B16F10的抑瘤率较iNGR-EPAPKP-IAE-1提高了22.02%,较IAE-1+iNGR抑瘤率提高了26.80%,较IAE-1-GGGGS(G4S)-iNGR抑瘤率提高了16.81%,较IAE-1-(GS)5-iNGR抑瘤率提高了15.78%,较IAE-1-EPAPK-iNGR抑瘤率提高了11.88%;IAE-1-EPAPKP-iNGR对4T1的抑瘤率较iNGR-EPAPKP-IAE-1提高了32.4%,较IAE-1+iNGR抑瘤率提高了31.54%,较IAE-1-GGGGS(G4S)-iNGR抑瘤率提高了26.43%,较IAE-1-(GS)5-iNGR抑瘤率提高了23.51%,较IAE-1-EPAPK-iNGR抑瘤率提高了22.53%。这些对比数据证明了:相对于使用其他linker而言,使用EPAPKP作为Linker时组成的融合蛋白能够更好的发挥抑瘤作用;此外,相对于将iNGR连接到超抗原的N端而言,将iNGR连接到超抗原的C端能使融合蛋白更好的发挥抑瘤作用。
(4)为了对比本发明中的IAE-1、IAE-2、IAE-3与其他抗肿瘤效应分子(包括其他超级抗原)连接同样的连接肽和iNGR后的抗肿瘤效果的差异,构建了以同样连接方式连接不同效应分子所组成的四种融合蛋白SEA-EPAPKP-iNGR、SEB-EPAPKP-iNGR、SEC2-EPAPKP-iNGR、sTRAIL-EPAPKP-iNGR,并且对比了其生物学活性。实验步骤同实施例5。
结果发现以SEA和SEB构建的融合蛋白SEA-EPAPKP-iNGR、SEB-EPAPKP-iNGR的毒性很强,动物实验中很快导致动物死亡(图9、图10);以SEC2构建的融合蛋白SEC2-EPAPKP-iNGR在同样剂量下抑瘤效果不如改构体IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR(图9、图10)。
选择分子量相近的融合蛋白sTRAIL-EPAPKP-iNGR的抑瘤效果进行对比,实验方法同实施例4,结果如图5和图6显示在相同浓度(350pmol/μl)下,IAE-1-EPAPKP-iNGR对B16F10的抑瘤率较sTRAIL-EPAPKP-iNGR高47.98%,对4T1的抑瘤率较sTRAIL-EPAPKP-iNGR高53%。说明了融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR的这种组合方式更换为其他类型的效应分子如sTRAIL-EPAPKP-iNGR后,不能表现出同样理想的效果。
将效应分子替换为超抗原SEA、SEB、SEC2,并以相同的方式连接iNGR组成的融合蛋白SEA-EPAPKP-iNGR、SEB-EPAPKP-iNGR、SEC2-EPAPKP-iNGR,按照同实施例5一致的实验方法获得的小鼠生存曲线如图9(野生SEC2对SEA的P=0,野生SEC2对SEB的P=0.001)和图10(野生SEC2对SEA的P=0.001,野生SEC2对SEB的P=0.001)所示,图中显示,融合蛋白SEA-EPAPKP-iNGR给药组B16F10造模小鼠的生存中位数为11天,融合蛋白SEB-EPAPKP-iNGR给药组B16F10造模小鼠的平均生存天数为12天,低于对照组(PBS)的平均生存天数(18.5天),而融合蛋白SEC2-EPAPKP-iNGR给药组B16F10造模小鼠的平均生存天数为20.5天,融合蛋白IAE-1-EPAPKP-iNGR的平均生存天数为29.5天,IAE-2-EPAPKP-iNGR的平均生存天数为28.5天,IAE-3-EPAPKP-iNGR的平均生存天数为29.5天。融合蛋白SEA-EPAPKP-iNGR给药组4T1造模小鼠的生存中位数为26.5天,融合蛋白SEB-EPAPKP-iNGR给药组4T1造模小鼠的平均生存天数为29天,低于对照组(PBS)的平均生存天数(34天),而融合蛋白SEC2-EPAPKP-iNGR给药组4T1造模小鼠的平均生存天数为48天,融合蛋白IAE-1-EPAPKP-iNGR的平均生存天数为56.5天,IAE-2-EPAPKP-iNGR的平均生存天数为59.5天,IAE-3-EPAPKP-iNGR的平均生存天数为61天。这一结果显示融合蛋白IAE-1-EPAPKP-iNGR、IAE-2-EPAPKP-iNGR、IAE-3-EPAPKP-iNGR所表现出的理想的抑瘤效果,在其他超抗原或者非超抗原类的效应分子上没有普遍性。
SEQUENCE LISTING
<110> 中国科学院沈阳应用生态研究所
<120> 一种融合蛋白及其制备与应用
<130> P20016581C
<160> 34
<170> PatentIn version 3.5
<210> 1
<211> 762
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPKP-iNGR基因iae-1-epapkp-ingr碱基序列
<400> 1
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa agctgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aaccttgccg caacggccgc ggcccggatt gc 762
<210> 2
<211> 254
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPKP-iNGR的氨基酸序列
<400> 2
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Pro Cys Arg Asn Gly Arg Gly Pro Asp Cys
245 250
<210> 3
<211> 239
<212> PRT
<213> Artificial Sequence
<220>
<223> IAE-1的氨基酸序列
<400> 3
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly
225 230 235
<210> 4
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> 连接短肽
<400> 4
Glu Pro Ala Pro Lys Pro
1 5
<210> 5
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> iNGR
<400> 5
Cys Arg Asn Gly Arg Gly Pro Asp Cys
1 5
<210> 6
<211> 762
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-2-EPAPKP-iNGR基因IAE-2-epapkp-iNGR碱基序列
<400> 6
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa acatgaagga 360
aacgcctttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aaccttgccg caacggccgc ggcccggatt gc 762
<210> 7
<211> 254
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-2-EPAPKP-iNGR的氨基酸序列
<400> 7
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys His Glu Gly Asn Ala Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Pro Cys Arg Asn Gly Arg Gly Pro Asp Cys
245 250
<210> 8
<211> 239
<212> PRT
<213> Artificial Sequence
<220>
<223> IAE-2的氨基酸序列
<400> 8
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys His Glu Gly Asn Ala Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly
225 230 235
<210> 9
<211> 762
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-3-EPAPKP-iNGR基因IAE-3-epapkp-iNGR碱基序列
<400> 9
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa agctgaagga 360
aacgcctttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aaccttgccg caacggccgc ggcccggatt gc 762
<210> 10
<211> 254
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-3-EPAPKP-iNGR的氨基酸序列
<400> 10
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn Ala Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Pro Cys Arg Asn Gly Arg Gly Pro Asp Cys
245 250
<210> 11
<211> 239
<212> PRT
<213> Artificial Sequence
<220>
<223> IAE-3的氨基酸序列
<400> 11
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn Ala Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly
225 230 235
<210> 12
<211> 813
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白SEA-EPAPKP-iNGR编码基因sea-epapkp-iNGR的碱基序列
<400> 12
aaaaaaacag catttacatt acttttattc attgccctaa cgttgacaac aagtccactt 60
gtaaatggta gcgagaaaag cgaagaaata aatgaaaaag atttgcgaaa aaagtctgaa 120
ttgcagggaa cagctttagg caatcttaaa caaatctatt attacaatga aaaagctaaa 180
actgaaaata aagagagtca cgatcaattt ttacagcata ctatattgtt taaaggcttt 240
tttacagatc attcgtggta taacgattta ttagtagatt ttgattcaaa ggatattgtt 300
gataaatata aagggaaaaa agtagacttg tatggtgctt attatggtta tcaatgtgcg 360
ggtggtacac caaacaaaac agcttgtatg tatggtggtg taacgttaca tgataataat 420
cgattgaccg aagagaaaaa agtgccgatc aatttatggc tagacggtaa acaaaataca 480
gtacctttgg aaacggttaa aacgaataag aaaaatgtaa ctgttcagga gttggatctt 540
caagcaagac gttatttaca ggaaaaatat aatttatata actctgatgt ttttgatggg 600
aaggttcaga ggggattaat cgtgtttcat acttctacag aaccttcggt taattacgat 660
ttatttggtg ctcaaggaca gtattcaaat acactattaa gaatatatag agataataaa 720
acgattaact ctgaaaacat gcatattgat atatatttat atacaagtga accggcgcca 780
aaaccttgcc gcaacggccg cggcccggat tgc 813
<210> 13
<211> 271
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白SEA-EPAPKP-iNGR的氨基酸序列
<400> 13
Lys Lys Thr Ala Phe Thr Leu Leu Leu Phe Ile Ala Leu Thr Leu Thr
1 5 10 15
Thr Ser Pro Leu Val Asn Gly Ser Glu Lys Ser Glu Glu Ile Asn Glu
20 25 30
Lys Asp Leu Arg Lys Lys Ser Glu Leu Gln Gly Thr Ala Leu Gly Asn
35 40 45
Leu Lys Gln Ile Tyr Tyr Tyr Asn Glu Lys Ala Lys Thr Glu Asn Lys
50 55 60
Glu Ser His Asp Gln Phe Leu Gln His Thr Ile Leu Phe Lys Gly Phe
65 70 75 80
Phe Thr Asp His Ser Trp Tyr Asn Asp Leu Leu Val Asp Phe Asp Ser
85 90 95
Lys Asp Ile Val Asp Lys Tyr Lys Gly Lys Lys Val Asp Leu Tyr Gly
100 105 110
Ala Tyr Tyr Gly Tyr Gln Cys Ala Gly Gly Thr Pro Asn Lys Thr Ala
115 120 125
Cys Met Tyr Gly Gly Val Thr Leu His Asp Asn Asn Arg Leu Thr Glu
130 135 140
Glu Lys Lys Val Pro Ile Asn Leu Trp Leu Asp Gly Lys Gln Asn Thr
145 150 155 160
Val Pro Leu Glu Thr Val Lys Thr Asn Lys Lys Asn Val Thr Val Gln
165 170 175
Glu Leu Asp Leu Gln Ala Arg Arg Tyr Leu Gln Glu Lys Tyr Asn Leu
180 185 190
Tyr Asn Ser Asp Val Phe Asp Gly Lys Val Gln Arg Gly Leu Ile Val
195 200 205
Phe His Thr Ser Thr Glu Pro Ser Val Asn Tyr Asp Leu Phe Gly Ala
210 215 220
Gln Gly Gln Tyr Ser Asn Thr Leu Leu Arg Ile Tyr Arg Asp Asn Lys
225 230 235 240
Thr Ile Asn Ser Glu Asn Met His Ile Asp Ile Tyr Leu Tyr Thr Ser
245 250 255
Glu Pro Ala Pro Lys Pro Cys Arg Asn Gly Arg Gly Pro Asp Cys
260 265 270
<210> 14
<211> 840
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白SEB-EPAPKP-iNGR编码基因seb-epapkp-iNGR的碱基序列
<400> 14
tataagagat tatttatttc acatgtaatt ttgatattcg cactgatatt agttatttct 60
acacccaacg ttttagcaga gagtcaacca gatcctaaac cagatgagtt gcacaaatcg 120
agtaaattca ctggtttgat ggaaaatatg aaagttttgt atgatgataa tcatgtatca 180
gcaataaacg ttaaatctat agatcaattt ctatactttg acttaatata ttctattaag 240
gacactaagt tagggaatta tgataatgtt cgagtcgaat ttaaaaacaa agatttagct 300
gataaataca aagataaata cgtagatgtg tttggagcta attattatta tcaatgttat 360
ttttctaaaa aaacgaatga tattaattcg catcaaactg acaaacgaaa aacttgtatg 420
tatggtggtg taactgagca taatggaaac caattagata aatatagaag tattactgtt 480
cgggtatttg aagatggtaa aaatttatta tcttttgacg tacaaactaa taagaaaaag 540
gtgactgctc aagaattaga ttacctaact cgtcactatt tggtgaaaaa taaaaaactc 600
tatgaattta acaactcgcc ttatgaaacg ggatatatta aatttataga aaatgagaat 660
agcttttggt atgacatgat gcctgcacca ggagataaat ttgaccaatc taaatattta 720
atgatgtaca atgacaataa aatggttgat tctaaagatg tgaagattga agtttatctt 780
acgacaaaga aaaaggaacc ggcgccaaaa ccttgccgca acggccgcgg cccggattgc 840
<210> 15
<211> 280
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白SEB-EPAPKP-iNGR的氨基酸序列
<400> 15
Tyr Lys Arg Leu Phe Ile Ser His Val Ile Leu Ile Phe Ala Leu Ile
1 5 10 15
Leu Val Ile Ser Thr Pro Asn Val Leu Ala Glu Ser Gln Pro Asp Pro
20 25 30
Lys Pro Asp Glu Leu His Lys Ser Ser Lys Phe Thr Gly Leu Met Glu
35 40 45
Asn Met Lys Val Leu Tyr Asp Asp Asn His Val Ser Ala Ile Asn Val
50 55 60
Lys Ser Ile Asp Gln Phe Leu Tyr Phe Asp Leu Ile Tyr Ser Ile Lys
65 70 75 80
Asp Thr Lys Leu Gly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys Asn
85 90 95
Lys Asp Leu Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe Gly
100 105 110
Ala Asn Tyr Tyr Tyr Gln Cys Tyr Phe Ser Lys Lys Thr Asn Asp Ile
115 120 125
Asn Ser His Gln Thr Asp Lys Arg Lys Thr Cys Met Tyr Gly Gly Val
130 135 140
Thr Glu His Asn Gly Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr Val
145 150 155 160
Arg Val Phe Glu Asp Gly Lys Asn Leu Leu Ser Phe Asp Val Gln Thr
165 170 175
Asn Lys Lys Lys Val Thr Ala Gln Glu Leu Asp Tyr Leu Thr Arg His
180 185 190
Tyr Leu Val Lys Asn Lys Lys Leu Tyr Glu Phe Asn Asn Ser Pro Tyr
195 200 205
Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Glu Asn Ser Phe Trp Tyr
210 215 220
Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr Leu
225 230 235 240
Met Met Tyr Asn Asp Asn Lys Met Val Asp Ser Lys Asp Val Lys Ile
245 250 255
Glu Val Tyr Leu Thr Thr Lys Lys Lys Glu Pro Ala Pro Lys Pro Cys
260 265 270
Arg Asn Gly Arg Gly Pro Asp Cys
275 280
<210> 16
<211> 762
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白SEC2-EPAPKP-iNGR编码基因sec2-epapkp-iNGR的碱基序列
<400> 16
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa acatgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aaccttgccg caacggccgc ggcccggatt gc 762
<210> 17
<211> 254
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白SEC2-EPAPKP-iNGR的氨基酸序列
<400> 17
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Pro Cys Arg Asn Gly Arg Gly Pro Asp Cys
245 250
<210> 18
<211> 239
<212> PRT
<213> Artificial Sequence
<220>
<223> SEC2氨基酸序列
<400> 18
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly
225 230 235
<210> 19
<211> 759
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-GGGGS(G4S)-iNGR编码基因IAE-1-ggggs-iNGR的碱基序列
<400> 19
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa agctgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggaggt 720
ggcggaggtt catgccgcaa cggccgcggc ccggattgc 759
<210> 20
<211> 253
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-GGGGS(G4S)-iNGR的氨基酸序列
<400> 20
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Gly
225 230 235 240
Gly Gly Gly Ser Cys Arg Asn Gly Arg Gly Pro Asp Cys
245 250
<210> 21
<211> 774
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-(GS)5-iNGR编码基因IAE-1-gsgsgsgsgs-iNGR的碱基序列
<400> 21
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa agctgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggaggt 720
tcaggctccg gaagcggttc aggttcctgc cgcaacggcc gcggcccgga ttgc 774
<210> 22
<211> 258
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-(GS)5-iNGR的氨基酸序列
<400> 22
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Ser Gly Ser Cys Arg Asn Gly Arg Gly Pro
245 250 255
Asp Cys
<210> 23
<211> 759
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPK-iNGR编码基因IAE-1-epapk-iNGR的碱基序列
<400> 23
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa agctgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aatgccgcaa cggccgcggc ccggattgc 759
<210> 24
<211> 253
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPK-iNGR的氨基酸序列
<400> 24
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Cys Arg Asn Gly Arg Gly Pro Asp Cys
245 250
<210> 25
<211> 762
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白iNGR-EPAPKP-IAE-1编码基因iNGR-epapkp-IAE-1的碱基序列
<400> 25
tgccgcaacg gccgcggccc ggattgcgaa ccggcgccaa aacctgagag tcaaccagac 60
cctacgccag atgagttgca caaatcaagt gagtttactg gtttgatgga aaatatgaaa 120
tatttatatg atgatcatta tgtatcagca actaaagtta tgtctgtaga taaatttttg 180
gcacatgatt taatttataa cattagtgat aaaaaactaa aaaattatga caaagtgaaa 240
acagagttat taaatgaaga tttagcaaag aagtacaaag atgaagtagt tgatgtgtat 300
ggatcaaatt actatgtaaa ctgctatttt tcatccaaag ataatgtagg taaagttaca 360
ggtggtaaaa cttgtatgta tggaggaata acaaaagctg aaggaaacca ctttgataat 420
gggaacttac aaaatgtact tataagagtt tatgaaaata aaagaaacac aatttctttt 480
gaagtgcaaa ctgataagaa aagtgtaaca gctcaagaac tagacataaa agctaggaat 540
tttttaatta ataaaaaaaa tttgtatgag tttaacagtt caccatatga aacaggatat 600
ataaaattta ttgaaaataa cggcaatact ttttggtatg atatgatgcc tgcaccaggc 660
gataagtttg accaatctaa atatttaatg atgtacaacg acaataaaac ggttgattct 720
aaaagtgtga agatagaagt ccaccttaca acaaagaatg ga 762
<210> 26
<211> 254
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白iNGR-EPAPKP-IAE-1的氨基酸序列
<400> 26
Cys Arg Asn Gly Arg Gly Pro Asp Cys Glu Pro Ala Pro Lys Pro Glu
1 5 10 15
Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu Phe
20 25 30
Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr Val
35 40 45
Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp Leu
50 55 60
Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val Lys
65 70 75 80
Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu Val
85 90 95
Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser Ser
100 105 110
Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr Gly
115 120 125
Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln
130 135 140
Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe
145 150 155 160
Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile
165 170 175
Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn
180 185 190
Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly
195 200 205
Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp
210 215 220
Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser
225 230 235 240
Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly
245 250
<210> 27
<211> 744
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPKP-NGR编码基因IAE-1-epapkp-NGR的碱基序列
<400> 27
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa agctgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aacctaacgg ccgc 744
<210> 28
<211> 248
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPKP-NGR的氨基酸序列
<400> 28
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Pro Asn Gly Arg
245
<210> 29
<211> 756
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPKP-tLyp-1编码基因IAE-1-epapkp-tlyp-1的碱基序列
<400> 29
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtttg 60
atggaaaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa agctgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aaccttgcgg caacaaacgc acccgt 756
<210> 30
<211> 252
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白IAE-1-EPAPKP-tLyp-1的氨基酸序列
<400> 30
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Leu Met Glu Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys Ala Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Pro Cys Gly Asn Lys Arg Thr Arg
245 250
<210> 31
<211> 549
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白sTRAIL-EPAPKP-iNGR编码基因strail-epapkp-iNGR的碱基序列
<400> 31
gtgagagaaa gaggtcctca gagagtagca gctcacataa ctgggaccag aggaagaagc 60
aacacattgt cttctccaaa ctccaagaat gaaaaggctc tgggccgcaa aataaactcc 120
tgggaatcat caaggagtgg gcattcattc ctgagcaact tgcacttgag gaatggtgaa 180
ctggtcatcc atgaaaaagg gttttactac atctattccc aaacatactt tcgatttcag 240
gaggaaataa aagaaaacac aaagaacgac aaacaaatgg tccaatatat ttacaaatac 300
acaagttatc ctgaccctat attgttgatg aaaagtgcta gaaatagttg ttggtctaaa 360
gatgcagaat atggactcta ttccatctat caagggggaa tatttgagct taaggaaaat 420
gacagaattt ttgtttctgt aacaaatgag cacttgatag acatggacca tgaagccagt 480
tttttcgggg cctttttagt tggcgaaccg gcgccaaaac cttgccgcaa cggccgcggc 540
ccggattgc 549
<210> 32
<211> 183
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白sTRAIL-EPAPKP-iNGR的氨基酸序列
<400> 32
Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr
1 5 10 15
Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys
20 25 30
Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His
35 40 45
Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His
50 55 60
Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln
65 70 75 80
Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr
85 90 95
Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser
100 105 110
Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser
115 120 125
Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe
130 135 140
Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser
145 150 155 160
Phe Phe Gly Ala Phe Leu Val Gly Glu Pro Ala Pro Lys Pro Cys Arg
165 170 175
Asn Gly Arg Gly Pro Asp Cys
180
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> 正向引物
<400> 33
cggaattcga gagtcaacca gaccc 25
<210> 34
<211> 86
<212> DNA
<213> Artificial Sequence
<220>
<223> 反向引物
<400> 34
ccctcgagtt agcaatccgg gccgcggccg ttgcggcaag gttttggcgc cggttctcca 60
ttctttgttg taaggtggac ttctat 86
Claims (10)
1.一种融合蛋白,其特征在于,所述融合蛋白从N端到C端依次包括:SEC2突变体、连接短肽和iNGR,
所述SEC2突变体在如SEQ ID NO:18所示序列的第20、22、118和/或122位氨基酸残基发生突变,
所述连接短肽的氨基酸序列如SEQ ID NO:4所示,
所述iNGR的氨基酸序列如SEQ ID NO:5所示。
2.如权利要求1所述的融合蛋白,其特征在于,所述SEC2突变体在如SEQ ID NO:18所示序列上具有T20L、G22E、H118A、H122A中的4、3、2或1个氨基酸取代;
较佳地,所述SEC2突变体的氨基酸序列如SEQ ID NO:3、SEQ ID NO:8或SEQ ID NO:11所示。
3.如权利要求1或2所述的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如序列表中SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:10所示;
较佳地,编码所述融合蛋白的核苷酸序列如序列表中SEQ ID NO:1、SEQ ID NO:6、SEQID NO:9所示。
4.一种融合基因,其特征在于,其编码如权利要求1或2所述的融合蛋白;
较佳地,其核苷酸序列如序列表中SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:9所示。
5.一种重组表达载体,其特征在于,所述重组表达载体中含如权利要求4所述的融合基因;
较佳地,所述重组表达载体的骨架载体为pET-28a-TEV。
6.一种转化体,其特征在于,其通过在宿主中导入如权利要求4所述的融合基因或者如权利要求5所述的重组表达载体即得;
较佳地,所述宿主为大肠杆菌,优选为大肠杆菌E.coliBL21(DE3)细胞或者E.coliTG1。
7.一种融合蛋白的制备方法,其包括以下步骤:(1)获得如权利要求6所述的转化体;(2)筛选所述转化体,表达并纯化所述融合蛋白。
8.如权利要求7所述的制备方法,其特征在于,步骤(2)中,所述纯化包括将所述表达所得的菌体经超声波破碎后离心收集上清液,经过两次Ni亲合层析即可;
较佳地,所述Ni亲合层析包括以0.2-0.8ml/min的上样速度将样品上样于预先平衡好的Ni亲合层析柱;使用8-12个柱体积的平衡缓冲液洗涤后用洗脱缓冲液洗脱即可,优选使用10个柱体积的平衡缓冲液洗涤;
更佳地,所述平衡缓冲液为含有20-80mM咪唑的平衡缓冲液,其组成优选为:20-30mMTirs-HCl,800-1000mM NaCl,20-80mM咪唑,和/或,所述平衡缓冲液的pH值为7.2~8.0;和/或,所述洗脱缓冲液为含有250-300mM咪唑的洗脱缓冲液,其组成优选为:20-30mM Tirs-HCl,800-1000mM NaCl,250-300mM咪唑;和/或,所述洗脱缓冲液的pH值为7.2~8.0。
9.如权利要求8所述的制备方法,其特征在于,在所述的两次Ni亲合层析之间还包括超滤脱盐的步骤;
较佳地,在所述超滤脱盐后还包括与TEV蛋白酶混合酶切的步骤;更佳地,所述超滤脱盐后的产物与所述TEV蛋白酶的摩尔比为1:5;和/或,所述酶切的时间为24h。
10.一种如权利要求1~3任一项所述的融合蛋白、如权利要求4所述的融合基因、如权利要求5所述的重组表达载体、或如权利要求6所述的转化体在制备药物中的应用;优选在制备治疗肿瘤的药物中的应用,更优选在制备治疗肿瘤免疫的药物中的应用。
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CN102060916A (zh) * | 2010-09-08 | 2011-05-18 | 沈阳协合生物制药股份有限公司 | 肠毒素c2超抗原突变蛋白和编码基因及其制备和应用 |
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