CN1768862A - 一类能与表达GnRH受体的癌细胞特异结合且用锝－99m标记的蛋白 - Google Patents
一类能与表达GnRH受体的癌细胞特异结合且用锝－99m标记的蛋白 Download PDFInfo
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Abstract
本发明是通过将能特异性识别表达GnRH受体的瘤细胞的一类蛋白用锝－99m标记，从而产生一种能在人体内通过闪烁成像，检测体内瘤细胞的表面受体类型及瘤位置的蛋白类体内检测剂。此类蛋白主要是天然GnRH，GnRH突变体，以及由它们与PE毒素的衍生物组成的导向融合蛋白，现今主要是被开发利用为靶向毒素物质，去特异性杀伤部分表达GnRH受体的癌细胞。
Description
一.技术领域
目前癌症仍然是严重威胁人类健康的疾病，发病率和死亡率都很高。癌症的传统治疗方法主要有3种方法：化疗，放疗和手术治疗。手术疗法仅限于早期，而化疗和放疗虽然有效，但是其严重的毒副作用限制了这种方法不能使用大剂量和长期应用，从而大大影响了其治疗效果。
因此，现阶段人们致力于研究一种低毒高效治疗肿瘤的新方法，就是消除放、化疗方法在杀伤肿瘤细胞的同时也大量杀伤正常细胞的缺点，特异性地针对肿瘤细胞进行杀伤，而对正常细胞低毒性或没有毒性。这种方法就是应用靶向分子，包括肿瘤细胞表面过量表达的受体的配体和单克隆抗体等，这种靶向分子进入体内可以特异性地到达肿瘤部位，而与其相连的效应蛋白或与其偶联的化疗药物可以在肿瘤部位杀伤肿瘤细胞，从而达到特异性治疗肿瘤的目的。
但是，由于该类融合蛋白只对表达靶向分子受体的癌细胞起作用，使得在给药前能清楚了解目的癌细胞是否表达靶向分子受体，变得非常重要。这样可以大大提高治疗效果，避免耽误无关病人的治疗。
二.背景技术
靶向性药物的研究是癌症治疗的一大进步，目前，人们发现约40％的癌细胞表面特异性表达GnRH受体，故而针对性研究以GnRH靶向物的靶向融合蛋白，去特异性的杀死癌细胞，具有跨时代的意义。然而，在用该类药物治疗前，能明确知道病人所患的癌症是否表达GnRH受体，就变得尤为重要。使得该类药物的靶向性效果更强.此类靶向药物基本结构为：导向部分通常连接一个毒素蛋白，如假单胞菌外毒素(PE)，白喉毒素(DT)，蓖麻毒素等，现在较常用的是假单胞菌外毒素A(PE)，PE是由假单胞杆菌分泌的，由613个氨基酸组成，其分为3个区域，I区为动物细胞受体识别区，通过此区与动物细胞相结合，II区为跨膜区，通过此区毒素分子进入细胞，III区为活性区，通过对细胞蛋白合成过程中的延伸因子进行核糖基化，使蛋白合成终止，从而导致细胞的死亡(Allured.V etc.Proc Natl Acad Sci.1986，83，1320-1324)。通过删除N末端1-252个氨基酸后就得到了截短的PE分子，剩下从N末端253-613的一段，为PE40，在去掉I区后，失去了动物细胞受体结合区，因为PE分子的细胞毒性作用发挥的前提是PE分子必须进入细胞内，所以PE40分子对于正常细胞基本没有毒性，由此PE40分子同各种导向分子连接，就构成了各种靶向毒素，应用于肿瘤的治疗以及其他疾病。通过缺失PE分子1-252和365-380两段序列，剩下的253-364和381-631两段序列按原顺序相连，就得到PE38分子。通过缺失PE分子的1-279位氨基酸，剩下280-613部分，就得到了PE37分子。通过缺失PE分子的1-279位和365-380位两段序列，剩下280-364与381-631部分，就得到了PE35分子(Theuer CP etc.J Biol Chem.1992，267，(24)：16872-7)。PE35，PE37，PE38和PE40都是PE分子的各种衍生物。
在核医学领域，通过探测少量体内给药的放射性标记示踪化合物(称放射性示踪物或放射性药物)的分布，确定病理状态的位置或测定其范围。探测这些放射性药物的方法通常被称作成像方法或放射成像方法。在放射成像中，放射标记是一种发射γ-射线的放射性核素，用一个γ-射线探测照相机确定该放射踪的位置，此方法通常被称作γ闪烁照相法。在许多情况下，用一种放射性标记的特异性结合化合物作为一种放射性药物具有特别的意义，此放射性药物具体确定体内的病理位置。
已知许多放射性核素可用放射性成像，包括：67Ga、99mTc(Tc-99m)、111In、123I、125I、109Yb、或186Re。要获得人体最理想的放射性成像必须考虑许多因素。为了最大限度地提高探测效果，优选一种放射性核素，其发射出的γ-射线能量在100-200Kev范围内。为减少病人吸收的辐射量，放射性核素的物理半衰期应同成像过程所要求的一样短。为使检查可以在任何日期及该日期的任何时间进行，在临床场所最好拥有一个总能得到的放射性核素源，Tc-99m是一种优选的放射性核素，因为它释放的γ-射线为140KeV，它的物理半衰期为6小时。并且一台钼一99/锝一99m发生器在临床现场容易获得。现有技术中所用的其它放射形核素，其优点均不如Tc-99m，这是因为这些放射性核素的物理半衰期较长，导致病人所吸收的辐射量较多(例如铟-111)。或者，别的一些放射性核素的辐射能量明显低于(例如碘-125)或明显高于(例如碘-131)Tc-99m，因而不适合闪烁照相的成像的质量要求，另外，有很多不适宜的放射性核素不能用现场发生器产生。
而且，用Tc-99m标记化合物与蛋白质，世界上已有广泛应用。
Byrne et al.，美国专利4,434,151中描述了高半胱氨酸硫代内酯衍生的双管能合剂，他能够将放射性核素偶合到含有氨基的化合物上，此化合物能定位在一个将被成像的器官或组织中。
Byrne et al.，美国专利4,571,556描述了新的用于螯合放射性核素的高半胱氨酸硫代内酯双官能螯合剂，它能将放射性核素耦合到末端含有氨基的化合物上，此化合物能定位于将被成像的器官或组织中。
Kondo et al.，欧洲专利申请，公开号483704A，公开了一种用一种基于甘氨酸-甘氨酸-甘氨酸部分制备一种Tc-99m螯合物的方法。
欧洲专利申请84109831.2描述了二酰氨基，二硫羟Tc-99m配位体及其盐用作为肾功能监测剂。
Albert et al UK专利申请8927255.3公开了使用111In标记的抑生长素衍生物(如奥曲肽)进行放射性成像，111In的标记是通过一个连接于氨基端的螯合基团完成。
Albert et al，国际专利申请Wo 91/01144公开了使用与生长因子、激素、干扰素和胞质分裂素有关的放射性标记肽进行放射性成像，而且这些放射性标记肽中含有一个与一个放射性核素螯合基团以共价键键合的特异性识别肽。
Kwekkeboom et al，1991，J Nucl.Med.32：981，Abstract#305涉及带有111In的放射性标记抑生长素的类似物。
Albert et al，1991，Abstract LM 10，12th American Peptide Symposium：1991描述了111In标记的二亚乙基三胺五乙酸衍生的抑生长素类似物的使用。
Cox et al，1991，Abstract，7th International Symposium on Radiopharmacology，P，16公开了Tc-99m-，131I-，以及111In-标记的抑生长素类似物在体内内分泌肿瘤闪烁法放射性定位中的用途。
Hnatowich，美国专利4,668,503描述了Tc-99m蛋白放射性标记。
Tolman，美国专利4,732,684描述了靶分子与金属硫蛋白片段的共轭。
Nicolotti et al，美国专利4,861,869描述了用于与生物分子(如抗体)形成共轭物的双官能偶联剂。
Fritzberg et al，美国专利4,965,392描述了用于标记蛋白的多种基于S-保护的巯基乙酰甘氨酰甘氨酸的螯合剂。
Schochat et al，美国专利5,061,641公开了对蛋白进行直接放射性标记，这种蛋白至少含有一个“侧”巯基。
Gustavson et al，美国专利5,112,953描述了用于放射性标记蛋白的Tc-99m螯合剂。
Dean et al，美国专利5,180,816描述了放射性标记一种蛋白的方法，它是用Tc-99m通过一种双官能螯合剂完成的。
Goedemans et al，PCT申请Wo 89/07456描述了用环状硫羟化合物，特别是2-亚氨基硫羟烷及其衍生物，放射性标记蛋白。
Dean et al，国际专利申请，公开号Wo 89/12625中讲述了用于Tc-99m蛋白标记的双官能偶联剂。
Schoemaker et al，国际专利申请，公开号Wo 89/06323中公开了一类嵌合蛋白，此蛋白含一个结合金属的区域。
Rhodes，1974，Sem.Nucl，Med.4：281-293讲述了用锝-99m标记人体血清白蛋白.
Khaw et al，1982，J.Nucl，Med.23；1011-1019公开了用Tc-99m标记具有生物活性的大分子的方法.
标记肽的尝试现在技术中有过报道.
Ege et al，美国专利4,832,940讲述了用于成像定位的T-淋巴细胞的放射性标记肽.
Morgan et al，美国专利4,986,979公开了成像感染部位的方法.
Lees et al，1989，PCT/US 89/01854讲述了用于动脉造影的放射性标记肽.
Morgan et al，国际专利申请，公开号WO 90/10463公开了成像感染部位的方法.
Cox，国际专利申请PCT/US 92/04559公开了含有二个半胱氨酸残基的放射性标记的抑生长素衍生物。
三.发明内容：
本发明是通过将能特异性识别表达GnRH受体的瘤细胞的一类蛋白用锝-99m标记，从而产生一种能在人体内通过闪烁成像，检测体内瘤细胞的表面受体类型及瘤位置的蛋白类体内检测剂。此类蛋白主要是天然GnRH，GnRH突变体，以及由它们与PE毒素的衍生物组成的形成的导向融合蛋白，现今主要是被开发利用为靶向毒素物质，去特异性杀伤部分表达GnRH受体的癌细胞。
本发明就是针对为该类靶向药物找寻靶点，从而在GnRH导向剂或GnRH-PE融合蛋白上标记放射性核素，在用药前先注射入少量标记蛋白，通过放射成像，检测癌症病人的癌细胞是否对靶向物具有特异吸引性，从而判断该类癌细胞是否对此类靶向药物敏感，以及癌细胞在体内的分布，便于该类靶向杀癌药物的有的放矢。
首先，在实例中我们将锝-99m标记的目标蛋白注射到肿瘤模型动物体内，用放射成像技术可清楚观察到肿瘤块的亮斑。其次，锝-99m体内半衰期仅6小时，且可以在现场用一台发生器制得，所以，选用锝-99m标记目的蛋白使检测更为安全，快捷。再者，正常体内GnRH受体主要分布在脑垂体，其他正常组织较少分布，而融合蛋白分子量较大，不能透过血脑屏障，不能影响脑垂体功能，对机体不产生不良反应，安全性较高。所以用锝-99m标记的融合蛋白诊断表达GnRH受体的肿瘤的部位及其对药物敏感性是确实可行的方法，也可进一步提高临床肿瘤的治疗水平。
故而，本发明的重点在于靶GnRH及其突变体以及靶GnRH作为导向的靶向融合蛋白标记上锝，形成的一种能在体内探测GnRH受体特异表达的癌组织物质，将为人类癌症的治疗作为相应的贡献。
实例：1
以基因工程手段与方法克隆出含GnRH蛋白基因的重组表达质粒。将该质粒转化到工程菌中后，经表达、纯化得到纯度较高的GnRH蛋白样品，再在体外对GnRH进行Tc-99m的放射标记，标记好的蛋白经除菌处理后静脉注射到荷瘤小鼠体内。注射样品2-4小时后进行闪烁成像，观测到肿瘤块所处的位置。
结果如图1，
实例：2
以基因工程手段与方法克隆出含GnRH-PE40融合蛋白基因的重组表达质粒。将该质粒转化到工程菌中后，经表达、纯化得到纯度较高的GnRH-PE40融合蛋白样品，再在体外对GnRH-PE40融合蛋白进行Tc-99m的放射标记，标记好的蛋白经除菌处理后静脉注射到荷瘤小白鼠体内。注射样品2-4小时后进行闪烁成像，观测到肿瘤块所处的位置。
结果如图2，
实例：3
以基因工程手段与方法克隆出含GnRH-PE40KDEL融合蛋白基因的重组表达质粒。将该质粒转化到工程菌中后，经表达、纯化得到纯度较高的GnRH-PE40KDEL融合蛋白样品，再在体外对GnRH-PE40KDEL融合蛋白进行Tc-99m的放射标记，标记好的蛋白经除菌处理后静脉注射到荷瘤小白鼠体内。注射样品2-4小时后进行闪烁成像，观测到肿瘤块所处的位置。
结果如图3，
实例：4
以基因工程手段与方法克隆出含GnRH-PE38KDEL融合蛋白基因的重组表达质粒。将该质粒转化到工程菌中后，经表达、纯化得到纯度较高的GnRH-PE38KDEL蛋白样品，再在体外对GnRH-PE38KDEL融合蛋白进行Tc-99m的放射标记，标记好的蛋白经除菌处理后静脉注射到荷瘤小白鼠体内。注射样品2-4小时后进行闪烁成像，观测到肿瘤块所处的位置。
结果如图4，
实例：5
以基因工程手段与方法克隆出含GnRH-PE37KDEL融合蛋白基因的重组表达质粒。将该质粒转化到工程菌中后，经表达、纯化得到纯度较高的GnRH-PE38KDEL融合蛋白样品，再在体外对GnRH-PE38KDEL蛋白进行Tc-99m的放射标记，标记好的蛋白经除菌处理后静脉注射到荷瘤小白鼠体内。注射样品2-4小时后进行闪烁成像，观测到肿瘤块所处的位置。
结果如图5，
实例：6
以基因工程手段与方法克隆出含GnRH-PE35KDEL融合蛋白基因的重组表达质粒。将该质粒转化到工程菌中后，经表达、纯化得到纯度较高的GnRH-PE35KDEL融合蛋白样品，再在体外对GnRH-PE35KDEL融合蛋白进行Tc-99m的放射标记，标记好的蛋白经除菌处理后静脉注射到荷瘤小自鼠体内。注射样品2-4小时后进行闪烁成像，观测到肿瘤块所处的位置。
结果如图6，
Claims (12)
1.一类能与表达GnRH受体的癌细胞特异结合且用锝-99m标记的蛋白，此类蛋白主要用于对表达GnRH受体的癌细胞，在人体内的检测及定位。其特征为：被锝-99m标记的这类蛋白一定包含有GnRH或者GnRH突变体的氨基酸序列。
2.如权力要求1所述，GnRH及其突变体的氨基酸序列如下：
Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly，其突变体为第六位的Gly(甘氨酸)被Lys(赖氨酸)，D-Lys，Ala(丙氨酸)，ornithine(鸟氨酸)，D-ornithine，Glu(谷氨酸)，D-Glu，Asp(天冬氨酸)，D-Asp，Cys(半胱氨酸)，D-Cys，Tyr(酪氨酸)和D-Tyr
3.如权力要求2所述，GnRH氨基酸序列的N末端多加一个蛋氨酸(Met)，使得该序列变为11个氨基酸。
4.GnRH-PE融合蛋白在组成结构上，GnRH与PE之间可以直接相连，也可以通过一段Linker相连。
5.如权力要求1，2，3所述，在GnRH及其突变体与PE40蛋白形成融合蛋白，该序列被锝-99m标记.
Met-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-TyrVal-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys
6.根据权利要求1，2，3所述，在GnRH及其突变体与PE40突变体蛋白形成融合蛋白，该序列被锝-99m标记：
Met-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Iyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
7.根据权利要求1，2，3所述，在GnRH及其突变体与PE38突变体蛋白形成融合蛋白，该序列被锝-99m标记：
Met-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-GIy-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
8.根据权利要求1，2，3所述，在GnRH及其突变体与PE37突变体蛋白形成融合蛋白，该序列被锝-99m标记
Met-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Ser-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
9.根据权利要求1，2，3所述，在GnRH及其突变体的N与PE37突变体蛋白形成融合蛋白，该序列被锝-99m标记
Met-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Ser-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Tbr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-ValArg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-His-Met-Ala-Glu-Glu-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Lys-Asp-Glu-Leu
10.根据权利要求1，2，3所述，在GnRH及其突变体与PE35突变体蛋白形成融合蛋白，该序列被锝-99m标记
Met-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Leu-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Leu-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu
11.权力要求5-9中所述的融合蛋白GnRH-PE，在PE组成上，包括天然的PE40，PE38，PE37，PE35，也包括PE蛋白的C末端5个氨基酸RDELK被序列KDEL替换了的PE突变体
12.权利要求1-10中所述，以上被锝标记的蛋白用于对表达GnRH受体的癌细胞，在人体内的检测及定位。
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| PCT/CN2005/001446 WO2006045233A1 (fr) | 2004-10-26 | 2005-09-09 | PROTÉINE MARQUÉE AU TECHNÉTIUM-99M ET COMPRENANT LA GnRH OU LA GnRH MUTANTE |
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| CN101195822B (zh) * | 2006-12-08 | 2012-08-08 | 湖南康都制药有限公司 | 高亲和性靶向融合蛋白 |
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| US5753206A (en) * | 1995-06-07 | 1998-05-19 | Immunomedics, Inc. | Radiometal-binding analogues of luteinizing hormone releasing hormone |
| US6140066A (en) * | 1998-03-24 | 2000-10-31 | Lorberboum-Galski; Haya | Methods of cancer diagnosis using a chimeric toxin |
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| CN101143897B (zh) * | 2007-09-27 | 2011-04-06 | 南京大学 | 99Tcm-His10-AnnexinV标记化合物的标记技术和作为显像剂在检测细胞凋亡方面的应用 |
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