CN1683416A - 高效低毒的系列功能蛋白 - Google Patents
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Abstract
本发明主要涉及一类由小分子肽与效应蛋白组成的系列融合蛋白，其具有特异杀灭过量表达该小分子肽受体细胞的特点，从而在多种疾病的治疗方面有潜在的应用价值。
Description
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。手术疗法仅限于早斯，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，并且这种方法治疗后容易产生耐药性肿瘤细胞，从而在复发的时候肿瘤变得无法控制。
因此，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电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。
背景技术
靶向性药物的研究是癌症治疗的一大进步，由于很多肿瘤细胞过量表达一些激素、细胞因子以及生长因子的受体，所以药物的导向部分可以应用激素类小分子，如人促黄体生成激素释放因子(GnRH)，生长因子，如EGF，细胞因子，如白细胞介素-2，白细胞介素-6等，电可以应用这些分子受体的单克隆抗体作为导向。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(TheuerCP etc.J Biol Chem.1992，267，(24)：16872-7)。PE37，PE38和PE40都是PE分子的各种衍生物。
发明内容
本发明是通过人促黄体激素释放因子(GnRH)与PE分子的各种衍生物通过一段Linker序列相连形成多种具有对肿瘤细胞具有导向性的融合蛋白。
权利要求书中的融合蛋白1是由GnRH与改进的PE40通过Linker相连得到，融合蛋白2是由GnRH与改进的PE38通过Linker相连得到，融合蛋白3是由GnRH与改进的PE37通过Linker相连得到，融合蛋白4是由改进的PE37与GnRH通过Linker相连得到。
GnRH的氨基酸序列为：Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly，文献表明，其第6位氨基酸Gly改变为D-Ala，可增强与受体的结合能力，本发明将GnRH的第6位的Gly突变为Ala，一定程度上提高了受体的结合能力。
我们发现，GnRH与PE衍生物直接相连后对肿瘤细胞的杀伤效率不高，这可能是由于GnRH的分子太小，只有10个氨基酸，有可能被PE衍生物分子包住而无法与细胞表面受体结合。常规选用的柔性Linker如Gly-Ser-Gly-Gly-Gly也不适合GnRH与PE衍生物的连接。由于上述的原因，GnRH分子很小，PE衍生物分子很大，所以用柔性的Linker PE衍生物分子可能会干扰GnRH与其受体的结合。本发明选用了序列His-Met-Ala-Glu-Glu作为Linker，该序列是一种相对刚性的Linker，能够有效地保证GnRH与PE衍生物两部分功能的实现，并且该Linker含有2个Glu，能有效地中和GnRH的碱性氨基酸，并且利于融合蛋白1，2，3分子在大肠杆菌中的高效表达。
PE衍生物的后5个氨基酸序列为Arg-Glu-Asp-Leu-Lys，本发明将其改为Lys-Asp-Glu-Leu，使其活性增强。
本发明的特点是通过突变的GnRH与改进的PE衍生物以及Linker之间的组合，而形成四种靶向性强的高效融合蛋白，具有表达量高，活性强等特点。
通过基因工程的方法，本发明的4种融合蛋白基因在大肠杆菌中得到了高效表达，通过常规的纯化方法得到了4种融合蛋白的纯品，其纯度在95％以上。
四.附图说明
图1是四种融合蛋白表达，发酵并纯化后的15％SDS-PAGE电泳图谱，A表示融合蛋白1，B表示融合蛋白2，C表示融合蛋白3，D表示融合蛋白4，E表示蛋白分子量标准，从上到下依次是97.4kD，66.2kD，43kD，31kD，20.1kD，14.4kD。
图2是融合蛋白细胞活性测定的曲线。
图3是融合蛋白细胞活性测定的细胞变化的显微照片。
五.具体实施方式
实例一.
根据Medline基因蛋白文库报道与相关文献报道，查出融合蛋白各部分天然蛋白序列与基因序列，再经过设计把两端天然蛋白经过Linker序列连接成完整的序列，然后根据设计的融合蛋白一级结构，用计算机排列出对应的基因序列，在排列出融合蛋白的表达基因时，根据不同表达体系宿主对不同氨基酸有偏好密码子的原则，分别排列出融合蛋白的原核序列与真核序列以便于在不同表达体系中进行高效表达。
以上述设计出的两条序列为蓝图，全基因合成2条完整的融合蛋白基因，经过测序确证后，将目的基因插入不同的表达载体，原核主要应用pET改进后系列质粒，真核系统主要采用pIC9K质粒系列，将确证后的重组子导入到宿主菌中，经过表达、发酵得到目的菌，然后通过机械方法破菌后，离心取上清，通过疏水层析和离子交换层析的方法纯化得到目的蛋白：融合蛋白1。得到的产物经过N末端分析，肽图分析以及质谱分析，确定为理论的融合蛋白1。最后进行细胞杀伤试验表明，融合蛋白1对Hela肿瘤细胞具有高特异性和高杀伤性。
测活方法(MTT法)：
首先对Hela细胞进行消化和收集，将细胞用RPMI 1640培养液稀释成6-8×104个/毫升的细胞悬液，接种于96孔板，100ul/孔，37度培养4小时。
样品先用RPMI 1640培养液稀释成100ul/mL，作为起始孔，然后按每孔与上孔2.5倍的关系稀释样品，各加入稀释的样品每孔100ul，37度，培养24小时，取出，用MTT法染色，用酶标仪570nm进行测定。对照加入100ul培养液。
实例二.
以实例一中全基因合成的2条基因序列为模板，利用PCR技术并合成多条引物，最后得到了对应于设计的融合蛋白2一级序列的目的基因序列片段，该片段经基因序列测定后插入表达质粒的表达框中，经检测插入正确后，把重组表达载体导入表达宿主菌中。该基因工程菌经过表达，发酵，纯化后得到了融合蛋白2，该蛋白经过实例1中的各项检测后确定其结构正确。
测活方法同实例一。
实例三.
以实例一中全基因合成的2条基因序列为模板，利用PCR技术并合成多条引物，最后得到了对应于设计的融合蛋白3一级序列的目的基因序列片段，该片段经基因序列测定后插入表达质粒的表达框中，经检测插入正确后，把重组表达载体导入表达宿主菌中。该基因工程菌经过表达，发酵，纯化后得到了融合蛋白3，该蛋白经过实例1中的各项检测后确定其结构正确。
测活方法同实例一。
实例四.
以实例一中全基因合成的2条基因序列为模板，利用PCR技术并合成多条引物，最后得到了对应于设计的融合蛋白4一级序列的目的基因序列片段，该片段经基因序列测定后插入表达质粒的表达框中，经检测插入正确后，把重组表达载体导入表达宿主菌中。该基因工程菌经过表达，发酵，纯化后得到了融合蛋白4，该蛋白经过实例1中的各项检测后确定其结构正确。
测活方法同实例一。
测活的结果：(每孔都重复一次，以下数据为2次的平均值)
从上表中及说明书附图2和附图3中可以看到，融合蛋白1-4对Hela肿瘤细胞具有很强的杀灭的作用。
Claims (7)
1.一种由小分子激素肽与效应蛋白组成的四种融合蛋白，分别为融合蛋白1，融合蛋白2和融合蛋白3和融合蛋白4。
2.根据权利要求1，所说的融合蛋白1的氨基酸序列为：
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-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-lle-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
3.根据权利要求1，所说的融合蛋白2的氨基酸序列为：
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-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-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
4根据权利要求1，所说的融合蛋白3序列为：
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-lle-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
5根据权利要求1，所说的融合蛋白4序列为：
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-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-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
6.权利要求1中，所说的四种融合蛋白使用的连接序列均为：His-Met-Ala-Glu-Glu。
7.权利要求1中的融合蛋白使用大肠杆菌、酵母或真核细胞为宿主菌，应用基因工程的方法进行表达、纯化获得。
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