WO2010022639A1

WO2010022639A1 - Target-specific double-mutant fusion protein

Info

Publication number: WO2010022639A1
Application number: PCT/CN2009/073441
Authority: WO
Inventors: 张俊英
Original assignee: 北京博翱泰生物技术有限公司
Priority date: 2008-08-25
Filing date: 2009-08-24
Publication date: 2010-03-04
Also published as: CN101343328A; CN101343328B

Abstract

The invention discloses a target specific double-mutant fusion protein, which is constructed from a fusion protein of human gonadotropin-releasing hormone mutant (mGnRH) and recombinant Pseudomonas aeruginosa exotoxin A mutant (PE38m4a). The mGnRH-PE38m4a protein of the present invention has significant specific binding activity and cytotoxicity to tumor cell lines including colon cancer HT-29 cells, ovarian cancer OVCAR3 cells, cervical adenocarcinoma HeLa cells and liver cancer HepG-2 cells, and the like; and has essentially no lethal effect to normal cells, and has a 9-10 fold higher activity than natural PEA, and is low-cost, easy to produce and suitable for industrial produce.

Description

靶特异性双突变体融合蛋白质技术领域 Target-specific double mutant fusion protein

本发明属生物技术领域，涉及具有靶细胞特异性细胞毒性的融合蛋白质，具体地说，本发明涉及人***释放激素突变体 (mGnRH)与重组绿脓杆菌外毒素 A突变体（PE38m4a)融合蛋白质的构建，以及所说的双突变体融合蛋白质作为抗肿瘤药物，在抑制类固醇激素剌激的肿瘤发生和发育中的应用。背景技术 The present invention belongs to the field of biotechnology and relates to a fusion protein having target cell-specific cytotoxicity. Specifically, the present invention relates to a human gonadotropin releasing hormone mutant (mGnRH) and a recombinant Pseudomonas aeruginosa exotoxin A mutant (PE38m4a). The construction of the fusion protein, and the double mutant fusion protein as an antitumor drug, inhibits the tumorigenesis and development of steroid hormone stimulation. Background technique

传统的肿瘤治疗方法是使用化学药物直接杀伤体内的肿瘤细胞。然而，大多数抗肿瘤药物在杀伤肿瘤的同时还杀伤正常细胞，即不可避免地造成严重的毒副作用。为了提高抗肿瘤药物对肿瘤细胞的选择性，自 20世纪 70年代后期以来，人们就试图将某些抗肿瘤药物或本来缺乏靶向选择性的毒素（包括细菌或动植物来源的毒素）化学偶联到适当的导向分子或称识别分子上，以制备杂交的具有靶特异性的抗肿瘤剂。随着分子生物学技术的发展，进而建立了以 DNA 重组技术制备这样的融合蛋白质的方法。 Traditional methods of cancer treatment use chemical drugs to directly kill tumor cells in the body. However, most anti-tumor drugs kill normal cells while killing tumors, which inevitably causes serious side effects. In order to improve the selectivity of anti-tumor drugs to tumor cells, since the late 1970s, people have tried to chemically block certain anti-tumor drugs or toxins that are targeted to selectively (including bacterial or animal-derived toxins). Linked to an appropriate targeting molecule or recognition molecule to prepare a hybrid target-specific anti-tumor agent. With the development of molecular biology techniques, a method for preparing such fusion proteins by DNA recombination technology was established.

在肿瘤的发生和发育过程中，许多肿瘤细胞都在其表面过量表达某些细胞因子或受体蛋白质，例如表皮生长因子受体（EGF-R)和转化生长因子 α受体（TGF ci -R)。因此，可将某些细胞毒性剂（如绿脓杆菌外毒素 (PE)、白喉毒素、霍乱毒素、葡萄球菌内毒素及蓖麻毒素等）连接到作为导向剂的 EGF、 TGF- α或 bFGF分子上，以产生兼有肿瘤细胞导向功能和细胞毒活性的杂交分子。这些杂交分子借助其对靶肿瘤细胞导向能力将整个分子引向靶细胞并通过其毒素部分杀伤靶细胞。 During tumorigenesis and development, many tumor cells overexpress certain cytokines or receptor proteins on their surface, such as epidermal growth factor receptor (EGF-R) and transforming growth factor alpha receptor (TGF ci-R). ). Therefore, certain cytotoxic agents (such as Pseudomonas aeruginosa exotoxin (PE), diphtheria toxin, cholera toxin, staphylococcal endotoxin, and ricin, etc.) can be linked to EGF, TGF-α or bFGF molecules as targeting agents. In order to produce a hybrid molecule having both tumor cell guiding function and cytotoxic activity. These hybrid molecules direct the entire molecule to the target cell by its ability to direct the target tumor cells and partially kill the target cell through its toxin.

目前研究较多且较深入的细胞毒性剂是绿脓杆菌外毒素 A (PEA) (参见美国专利 4， 545， 985)。 PEA是由 613个氨基酸组成的单链多肽。对 PE分子的 X射线晶体学研究和突变分析显示， PE 分子包括三个与产生细胞毒性相关的结构区域：负责与敏感细胞结合的氨基末端细胞受体结合区（I区）；负责毒素分子向细胞内转位的中间转位区（Π区）；以及负责失活细胞延伸因子 -2 并最终导致细胞死亡的羧基末端酶促活性区（III区）。其中 I 区包括介导细胞结合的 la区（氨基酸 1-252)和目前尚未明确其功能的 lb区（氨基酸 365-399)。可以使用生物化学或重组 DNA技术修饰 PE分子，以制备 PE分子中有一个或多个氨基酸缺失或取代的各种修饰的 PE片段，例如，一般将删去了 PE分子中 la区，只含有酶促和转位区，分子量约为 40KDa的 PE-A蛋白质称为 PE40。现已发现删除 PE的 la和大部分 lb区（氨基酸 365-380)后，即 PE38毒素分子，该分子仍保留其特异性细胞毒性，但降低了非特异性毒性和抗原性（例如参见 Hwang et al.， Ce l l 48： 129-136， 1987；美国专利 4， 892， 827和欧洲专利 0261671)。许多现有技术文献已描述了 PE与各种生长因子、抗体、激素或 CD4融合，以产生可选择性地导向并杀伤具有不同细胞膜蛋白（受体或抗原）之靶细胞的杂交蛋白质的方法（参见 Pastan and Fitz. G.， Science 254： 1173-1177, 1991)。例如， Chaudhary 等人（PNAS 84 : 4538-4542, 1987)描述了 PE40与 TGF α间形成的杂交体融合蛋白质（TGF α -ΡΕ40)。 TGF ci -PE40 的临床实用性依赖于其结合并杀伤具有表皮生长因子受体之细胞的能力。美国专利 5， 428， 143公开了用于选择性杀伤 HIV感染之细胞的杂交蛋白质及编码该杂交蛋白质的嵌合基因的构建。其中所说的杂交蛋白质由含有 HIV结合位点的人 CD4和可杀死 HI V感染之细胞毒性蛋白质（PE40)组成。 The more cytotoxic agent currently studied and more in-depth is Pseudomonas aeruginosa exotoxin A (PEA) (see U.S. Patent 4,545,985). PEA is a single-chain polypeptide consisting of 613 amino acids. X-ray crystallographic studies and mutational analysis of PE molecules revealed that the PE molecule includes three structural regions involved in the production of cytotoxicity: the amino terminal cell receptor binding region responsible for binding to sensitive cells (region I); responsible for toxin molecule orientation The intermediate translocation region (Π region) of intracellular translocation; and the carboxy-terminal enzymatically active region (region III) responsible for inactivating cell elongation factor-2 and ultimately leading to cell death. The region I includes the la region (amino acid 1-252) which mediates cell binding and the lb region (amino acid 365-399) which has not yet been clarified. The PE molecule can be modified using biochemical or recombinant DNA techniques to prepare various modified PE fragments having one or more amino acid deletions or substitutions in the PE molecule. For example, the LA region of the PE molecule is generally deleted, and only the enzyme is contained. In the promoting translocation region, the PE-A protein having a molecular weight of about 40 kDa is called PE40. It has been found that after removing the la of PE and most of the lb region (amino acid 365-380), the PE38 toxin molecule retains its specific cytotoxicity but reduces non-specific toxicity and antigenicity (see, for example, Hwang et al). Ce ll 48: 129-136, 1987; U.S. Patent 4,892,827 and European Patent 0 261 671. A number of prior art documents have described methods in which PE is fused to various growth factors, antibodies, hormones or CD4 to produce hybrid proteins that selectively direct and kill target cells having different cell membrane proteins (receptors or antigens) ( See Pastan and Fitz. G., Science 254: 1173-1177, 1991). For example, Chaudhary et al. (PNAS 84: 4538-4542, 1987) describe a hybrid fusion protein (TGF α -ΡΕ40) formed between PE40 and TGF α. The clinical utility of TGF ci-PE40 is dependent on its ability to bind and kill cells with epidermal growth factor receptors. U.S. Patent No. 5,428,143 discloses the construction of a hybrid protein for selectively killing HIV-infected cells and a chimeric gene encoding the hybrid protein. The hybrid protein described therein consists of human CD4 containing an HIV binding site and a cytotoxic protein (PE40) which kills HI V infection.

作为与本发明更为相关的现有技术，国际专利 W093/15751公开了将*** 释放激素 (GnRH)肽直接偶联到绿脓杆菌外毒素分子上制成的嵌合蛋白质分子。据称，投用这样的嵌合分子可导致脑垂体中携带 GnRH受体的细胞被破坏，并伴有性激素分泌的降低，因而可望将其用于动物不育化及抑制类固醇激素相关肿瘤的增殖。另外，国际专利申请 W097/15325描述了一种含有与绿脓杆菌外毒素化学结合的 GnRH的免疫原性载体***，以其作为疫苗可在动物体内诱发产生高浓度抗 GnRH抗体，因而可用于控制受孕、减少生殖激素驱动的行为及治疗类固醇激素反应性肿瘤。发明内容 As a prior art more relevant to the present invention, International Patent No. W093/15751 discloses a chimeric protein molecule prepared by directly coupling a gonadotropin releasing hormone (GnRH) peptide to a Pseudomonas aeruginosa exotoxin molecule. It has been reported that administration of such chimeric molecules can result in the destruction of cells carrying the GnRH receptor in the pituitary gland, accompanied by a decrease in the secretion of sex hormones, and thus it is expected to be used for animal sterility and inhibition of steroid-related tumors. proliferation. In addition, International Patent Application No. W097/15325 describes an immunogenic carrier system comprising GnRH chemically bound to Pseudomonas aeruginosa exotoxin, which can be used as a vaccine to induce high concentrations of anti-GnRH antibodies in animals and thus can be used for control Pregnancy, reduced reproductive hormone-driven behavior, and treatment of steroid-responsive tumors. Summary of the invention

本发明的目的是提供靶特异性双突变体融合蛋白质，它是由突变的*** 释放激素 mGnRH与重组绿脓杆菌外毒素 A突变体融合而成的融合毒素，特征在于所述的重组绿脓杆菌外毒素是去除 la 区及 lb 区的氨基酸 365-380，且 C 末端氨基酸 Glu610、 Leu612、 Lys613分别人工突变为 Lys610、 Glu612和 Leu613的 PEA分子，即 PE38m4a。 The object of the present invention is to provide a target-specific double mutant fusion protein which is a fusion toxin obtained by fusing a mutant gonadotropin releasing hormone mGnRH and a recombinant Pseudomonas aeruginosa exotoxin A mutant, characterized in that the recombinant green The exotoxin of Pseudomonas is the amino acid 365-380 which removes the la and lb regions, and the C-terminal amino acids Glu610, Leu612 and Lys613 are artificially mutated to the PEA molecules of Lys610, Glu612 and Leu613, respectively, ie, PE38m4a.

所述的突变的***释放激素 mGnRH,它的基因是以大肠杆菌偏性密码子为主体的基因形式合成的，氨基酸序列为： Met-Gly-G i u-Hi s-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-His (SEQ ID NO : 4)。 The mutant gonadotropin releasing hormone mGnRH, whose gene is synthesized by a gene encoding the Escherichia coli bias codon, and the amino acid sequence is: Met-Gly-G i u-Hi s-Trp-Ser- Tyr-Trp-Leu-Arg-Pro-Gly-His (SEQ ID NO: 4).

靶特异性双突变体融合蛋白质它的基因序列如序列表 SEQ ID NO : 1所示。 The target-specific double mutant fusion protein has the gene sequence as shown in the sequence listing SEQ ID NO: 1.

它的氨基酸序列如序列表 SEQ ID NO : 2所示。 Its amino acid sequence is shown in the Sequence Listing SEQ ID NO: 2.

一种表达载体， pET27-mGnRH-PE38m4a，其基因序列含有序列表 SEQ ID NO : 1 所示的序列。 An expression vector, pET27-mGnRH-PE38m4a, whose gene sequence contains the sequence shown in SEQ ID NO: 1.

本发明靶特异性双突变体融合蛋白质是由下述方法制备的； The target-specific double mutant fusion protein of the present invention is prepared by the following method;

(1)人工合成适合基因表达的 mGnRH基因序列，和 PE38m4a基因序列； (1) artificially synthesizing the mGnRH gene sequence suitable for gene expression, and the PE38m4a gene sequence;

(2)将人工合成的融合基因序列通过基因操作克隆到表达载体中； (2) cloning the artificially synthesized fusion gene sequence into an expression vector by genetic manipulation;

(3)用步骤 (2)的表达载体转化大肠杆菌中； (3) transforming E. coli with the expression vector of step (2);

(4)在适当条件下表达所说的融合蛋白质； (5)从细胞培养物中回收并纯化融合蛋白质。 (4) expressing said fusion protein under appropriate conditions; (5) The fusion protein is recovered and purified from the cell culture.

步骤 (l)mGnRH基因序列，采用人工设计合成了 Ncol 内切酶识别序列 --大肠杆菌偏性密码子组成的 mGnRH核苷酸序列，其中第 6位 Gly突变为 Trp—Ndel内切酶识别序列一PE38m4a核苷酸序列一EcoRI内切酶识别序列；合成的基因序列的 5 ' 和 3 ' 端分别引入了 Ncol和 EcoRI核酸内切酶酶切位点，并造成适于连接的粘性末端。 Step (l) The mGnRH gene sequence was artificially designed to synthesize the Ncol endonuclease recognition sequence, the mGnRH nucleotide sequence consisting of the E. coli bias codon, and the 6th Gly mutation was the Trp-Ndel endonuclease recognition sequence. A PE38m4a nucleotide sequence-EcoRI endonuclease recognition sequence; the 5' and 3' ends of the synthesized gene sequence respectively introduce Ncol and EcoRI endonuclease cleavage sites, and cause sticky ends suitable for ligation.

步骤 (2)所述的基因如序列表 SEQ ID NO : 1所示。 The gene described in the step (2) is shown in the sequence listing SEQ ID NO: 1.

所述的载体是 pET27。 The vector is pET27.

步骤 (3)所述的大肠杆菌为大肠杆菌 BL21 ( λ DE3)。 The Escherichia coli described in the step (3) is Escherichia coli BL21 (λ DE3).

本发明的再一个目的是靶特异性双突变体融合蛋白质在治疗与***释放激素受体有关的肿瘤药物的应用 A further object of the invention is the use of a target-specific double mutant fusion protein for the treatment of a tumor drug associated with a gonadotropin releasing hormone receptor

GnRH是由 Schal ly等于 1971年从动物体内分离纯化，阐明其结构后又人工合成的，并以此获得了 1976年的 Nobel奖。 GnRH为一个不含游离氨基酸与羧基的十肽，其分子结构为： P-Glu-Hi s-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO : 3)，其中第 4-6位氨基酸形成 β转折，呈发夹形，适合与受体结合，第 2和 3位对生物活性很重要，第 6位对维持发夹构象起重要作用，第 1位与第 4-10位氨基酸均参与受体结合，若置换以上氨基酸残基可导致活力丧失或呈几何级增强。 GnRH was isolated and purified from animals by Schally in 1971, and its structure was synthesized and then synthesized, and the Nobel Prize of 1976 was obtained. GnRH is a decapeptide containing no free amino acid and carboxyl group, and its molecular structure is: P-Glu-Hi s-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ (SEQ ID NO: 3) Among them, amino acids 4-6 form a β-transition, which is hairpin-shaped, which is suitable for binding to receptors. The second and third positions are important for biological activity, and the sixth position plays an important role in maintaining hairpin conformation. The 4th to 10th amino acids are involved in receptor binding, and replacement of the above amino acid residues may result in loss of vigor or geometrical enhancement.

在体内很容易被蛋白水解酶降解，故其半衰期仅 4-8min。其水解酶肽酶的主要作用部位是 Gly6-Leu7和 Pro9-GlylO_NH₂。为寻求高效且持久的 GnRH类似物，通过对其肽链结构中氨基酸的拾取或替换，已合成 3000多种 GnRH类似物。由于合成的 GnRH 半衰期长，作用更强，所以比天然的 GnRH更适于病人的治疗。合成长效的 GnRH激动剂的要求是稳定分子结构，使之不易被酶水解，增加与循环中的蛋白和胞膜的结合及提高对 GnRH受体的亲和力。如 6位为 D-氨基酸的类似物和取代 GlylO酰胺基。这种 GnRH激动剂不仅耐蛋白酶水解作用较大，而且对受体有较高的亲和力。若在第 6位引入庞大的疏水基团可进一步增加与受体的亲和力。这样的置换稳定了释放激素类似物的 "活性"构型，提高了与循环中蛋白的结合，从而延长半衰期。早期开发的 GnRH 拮抗剂的理论基础与 GnRH激动剂相似，可改善与受体结合，却产生不易接受的组胺释放副作用。因此，开发下一代的 GnRH拮抗剂的焦点集中到既提高效能又减少组胺释放这一方面。 It is easily degraded by proteolytic enzymes in the body, so its half-life is only 4-8min. The main sites of action of its hydrolase peptidase are Gly6-Leu7 and Pro9-GlylO_NH ₂ . To find efficient and long lasting GnRH analogs, more than 3,000 GnRH analogs have been synthesized by picking up or replacing amino acids in their peptide chain structures. Because the synthetic GnRH has a longer half-life and is more potent, it is more suitable for the treatment of patients than natural GnRH. The requirement for the synthesis of long-acting GnRH agonists is to stabilize the molecular structure, making it less susceptible to enzymatic hydrolysis, increasing binding to circulating proteins and membranes, and increasing affinity for the GnRH receptor. For example, the 6-position is an analog of D-amino acid and a substituted GlylO amide group. This GnRH agonist is not only resistant to protease hydrolysis but also has a high affinity for receptors. The introduction of a bulky hydrophobic group at position 6 further increases the affinity for the receptor. Such substitutions stabilize the "active" configuration of the release hormone analog, increasing binding to circulating proteins, thereby extending half-life. The theoretical basis for early development of GnRH antagonists is similar to that of GnRH agonists, which improves binding to receptors but produces unacceptable histamine release side effects. Therefore, the focus of developing the next generation of GnRH antagonists has focused on both improving efficacy and reducing histamine release.

越来越多的研究发现，某些激素和细胞因子的受体在肿瘤细胞上有异常高的表达，如 EGFR、 GnRHR等。而且，激素和细胞因子的分子相对较小，结构简单，基因操作方便，因此，作为免疫毒素的载体有很大的可行性。目前有很多的细胞因子和激素被利用来做免疫毒素的载体，如 IL-2、 IL-4、 EGF、 GnRH等，并且表达的重组免疫毒素蛋白均具有特异性细胞毒效应。 GnRH受体最早发现于垂体，随着近年来对 GnRH及其受体研究的进一步深入，越来越多的临床和实验表明，垂体外组织也有 GnRH及其受体的分布，并且垂体外正常组织及其相应部位癌组织 GnRH受体性质有许多不同。有文献报道，它们所介导的信号传导通路不同，也有报道，癌细胞表面的 GnRH受体可能介导细胞调亡。另外，癌细胞膜表面受体的亲和力通常大于相应正常组织细胞膜表面受体亲和力。据报导，在脊椎动物牛蛙脑垂体和后脑的细胞上已经发现了 3种 GnRH 受体的亚型，人体中目前发现两种亚型，但其分布和功能各不相同， I型受体与 I型 GnRH结合， II型受体与 II型 GnRH结合，二者之间交叉反应极低。已经有大量的实验证明：（1)人类正常组织的性腺（包括子宫内膜、子宫肌层、卵巢和睾丸），胎盘和大脑组织的细胞膜上有 GnRH受体的分布。（2)不排除其他正常组织细胞膜上有亲和力及低的 GnRH受体存在的可能，如肝脏。（3) GnRH受体主要分布在肝癌、胃癌、胰腺癌、结肠癌、卵巢癌、子宫内膜癌、子宫肌瘤、乳腺癌和***癌细胞膜上。 More and more studies have found that certain hormones and cytokine receptors have abnormally high expression on tumor cells, such as EGFR, GnRHR and so on. Moreover, hormones and cytokines have relatively small molecules, simple structure, and convenient gene manipulation. Therefore, as a carrier of immunotoxins, it is highly feasible. At present, many cytokines and hormones are used as carriers for immunotoxins, such as IL-2, IL-4, EGF, GnRH, etc., and the expressed recombinant immunotoxin proteins have specific cytotoxic effects. The GnRH receptor was first discovered in the pituitary. With the further study of GnRH and its receptors in recent years, more and more clinical and experimental studies have shown that GnRH and its The distribution of receptors, and the nature of GnRH receptors in normal tissues and their corresponding sites in the pituitary gland are many different. It has been reported in the literature that they signal different signaling pathways, and it has been reported that GnRH receptors on the surface of cancer cells may mediate apoptosis. In addition, the affinity of the cancer cell membrane surface receptor is generally greater than the corresponding normal tissue cell membrane surface receptor affinity. It has been reported that three subtypes of GnRH receptors have been found on the pituitary and hindbrain cells of vertebrate bullfrogs. Two subtypes are currently found in humans, but their distribution and function are different. Type I receptors and I Type GnRH binds, type II receptor binds to type II GnRH, and the cross-reaction between the two is extremely low. A large number of experiments have been carried out: (1) The distribution of GnRH receptors on the cell membrane of human placenta (including endometrium, myometrium, ovary and testis), placenta and brain tissue. (2) It is not excluded that there is affinity for other normal tissue membranes and the presence of low GnRH receptors, such as the liver. (3) GnRH receptors are mainly distributed on liver cancer, gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, endometrial cancer, uterine fibroids, breast cancer and prostate cancer cell membranes.

本文所使用的术语 "mGnRH" 是指能够与其表面上具有类固醇激素受体的细胞结合，并且当以高剂量投用于哺乳动物宿主时能引发抗 GnRH抗体产生的天然 GnRH的类似物或衍生物。 The term "mGnRH" as used herein refers to an analog or derivative of native GnRH capable of binding to a cell having a steroid hormone receptor on its surface and which elicits anti-GnRH antibody production when administered to a mammalian host at a high dose. .

天然 GnRH也称为促黄体激素释放激素（LHRH或 LRH)，其为具有下示氨基酸序列的十肽分子： Natural GnRH, also known as luteinizing hormone releasing hormone (LHRH or LRH), is a decapeptide molecule having the amino acid sequence shown below:

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO : 3)； pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO: 3);

式中所用的氨基酸符号为本领域通用的三字母缩写符号，并且其中的 pGlu代表焦谷氨酸。由于基因工程表达中无法合成焦谷氨酸，所以一般都将之突变为谷氨酰氨。可适用于本发明的 mGnRH类似物或衍生物是 Trp⁶-mGnRH。研究结果显示，分子中的第 3〜6位氨基酸是维持 GnRH活性所必需的，而 Ser⁴和 Tyr⁵则在受体结合中起到关键性作用。已有证据表明，卵巢细胞特别是粒层细胞表面具有 GnRH受体。已证明小鼠睾丸细胞（Legdig 细胞）表面的受体表达与体内激素水平有关（Harwood, J. P. et al.， Endocrinology 107 : 407-413, 1980) ₀ 另外，还有人发现大鼠子宫内膜上具有亲合力很高的 GnRH受体（曹咏清等，中国科学 B辑 1 : 32-37， 1984)。虽然目前对 GnRH 的垂体外作用机理尚不完全清楚，但可以肯定的是， GnRH 与性腺或子宫内膜等类固醇激素相关组织或细胞上特异性受体的结合是 GnRH 发挥垂体外作用的一个重要环节。而且也已显示，与靶细胞膜和细胞内分泌颗粒上存在的 GnRH受体结合基本上是没有种属特异性的（张崇理等，中国应用生理学杂志 5 (1) : 87-93， 1988)。 The amino acid symbols used in the formula are three-letter abbreviations commonly used in the art, and wherein pGlu represents pyroglutamic acid. Since pyroglutamic acid cannot be synthesized in genetic engineering expression, it is generally mutated to glutamine. An mGnRH analog or derivative that may be suitable for use in the present invention is Trp ⁶ -mGnRH. The results showed that amino acids 3 to 6 in the molecule are essential for maintaining GnRH activity, while Ser ⁴ and Tyr ⁵ play a key role in receptor binding. There is evidence that ovarian cells, particularly granulosa cells, have a GnRH receptor on their surface. Mouse testis cells (Legdig cells) hormone receptor expression has been demonstrated in vivo levels of the relevant surface (Harwood, JP et al, Endocrinology 107:. 407-413, 1980) 0 In addition, others have found that the rat endometrium GnRH receptor with high affinity (Cao Yuqing et al., Science in China B: 1: 32-37, 1984). Although the mechanism of action of GnRH in vitro is not fully understood, it is certain that the binding of GnRH to steroid hormone-related tissues such as gonads or endometrium or specific receptors on cells is an important factor in the role of GnRH in vitro. Link. It has also been shown that the binding to the GnRH receptor present on the target cell membrane and the endocrine granules is substantially species-specific (Zhang Chongli et al., Chinese Journal of Applied Physiology 5 (1): 87-93, 1988).

本发明所涉及的融合毒素中设计的导向部分为突变的 mGnRH 氨基酸序列为： Met-Gly-GI -Hi s-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-His (SEQ ID NO : 4) ; The guide portion designed in the fusion toxin of the present invention is a mutant mGnRH amino acid sequence: Met-Gly-GI-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-His (SEQ ID NO : 4) ;

式中所用的氨基酸符号为本领域通用的三字母缩写符号，其中为了基因连接方便在基因合成时于 N末端加入了 Met-Gly的密码子，并且将新序列中第八位氨基酸于基因水平上突变为 Trp，以使表达产物与相应受体结合能力的增强；同时将大肠杆菌偏性密码子引入到其中，以使表达产物适合在大肠杆菌中表达。我们对按本发明方法制备的 mGnRH-PE38m4a融合蛋白质的生物学活性分析已清楚地证明， mGnRH_PE38m4a 具有与包括 A549细胞在内多种性器官来源的肿瘤细胞具有杀伤活性，而对多种组织来源的正常细胞则没有这种特异杀伤活性（参见实施例 2)。 The amino acid symbols used in the formula are three-letter abbreviations commonly used in the art, in which a codon for Met-Gly is added to the N-terminus for gene synthesis, and the eighth amino acid of the new sequence is at the gene level. The mutation is Trp to enhance the ability of the expression product to bind to the corresponding receptor; and the E. coli bias codon is introduced therein to make the expression product suitable for expression in E. coli. We have a method according to the invention The biological activity analysis of the prepared mGnRH-PE38m4a fusion protein has clearly demonstrated that mGnRH_PE38m4a has cytotoxic activity against tumor cells derived from various sexual organs including A549 cells, but not for normal cells of various tissue sources. Killing activity (see Example 2).

本文中所使用术语 " IC₅。" 是指杀死靶细胞达到对照组的 50%所需的嵌合毒素的浓度。本发明中使用标准的 MTT法检测 " IC₅。 "值。 As used herein, the term "IC _5." Refers to kill the target cells reached a concentration of 50% in the control group chimeric toxin required. In the present invention, the "IC ₅ " value is detected using a standard MTT method.

Al lured等人对 PE晶体的 X线衍射分析表明 PE是 613个氨基酸组成的单链毒素蛋白，分子量 66kDa，其前体为 638个氨基酸，在分泌过程中切去了由 25个氨基酸组成的高度疏水的引导肽。从 PE 的晶体结构中得知，它在空间上分为三个结构域 (Domain, D)区，即 I区、 I I区和 I I I区（见图 1)。 I区在 PE的氨基端（N端），占分子的 1/3强，呈反向平行的 β 结构，它由 la区和 lb区组成，这两部分在 DNA序列上是分离的，但在三维结构中紧靠在一起。 la区由第 1〜252个氨基酸组成， lb区由第 365〜399个氨基酸组成。 I I 区由第 253〜364个氨基酸组成，为中央区，由 6_7个 a_螺旋组成。 I I I区由第 400〜613个氨基酸组成，为羧基端区，占分子的 1/3。此毒素的分子结构中有 8个半胱氨酸，形成 4个二硫键，它们是三级结构形成折叠的决定因素。 PEA结构功能的特点在于它的三个结构功能区在单一肽链上行使其细胞毒性所必需的细胞结合、转位和 ADP-核糖基化这三种功能，探知各区的功能及分析各区结构与功能的关系主要根据 PEA基因突变的研究。 X-ray diffraction analysis of PE crystal by Al lured et al. showed that PE is a single-stranded toxin protein of 613 amino acids with a molecular weight of 66 kDa, a precursor of 638 amino acids, and a height of 25 amino acids cut in the secretory process. Hydrophobic guide peptide. It is known from the crystal structure of PE that it is spatially divided into three domain (Down, D) regions, namely, I region, I I region, and I I I region (see Figure 1). The I region is at the amino terminus (N-terminus) of PE, which is 1/3 of the molecule, and has an antiparallel β structure. It consists of a la region and a lb region, which are separated in the DNA sequence, but in Close together in a three-dimensional structure. The la region consists of the first to 252 amino acids, and the lb region consists of the 365th to 399th amino acids. The I I region consists of 253 to 364 amino acids and is a central region composed of 6-7 a_helices. The I I I region consists of the 400th to 613th amino acids and is the carboxy terminal region, which accounts for 1/3 of the molecule. The toxin has eight cysteines in its molecular structure, forming four disulfide bonds, which are the determining factors for the formation of the tertiary structure. The structural function of PEA is characterized by its three structural functional regions, which are capable of cytotoxicity, such as cell binding, translocation and ADP-ribosylation, which are required for cytotoxicity in a single peptide chain, to ascertain the functions of each region and to analyze the structure of each region. The functional relationship is mainly based on the study of PEA gene mutations.

la区行使识别和结合靶细胞表面受体的功能， la区的缺失产生了分子量 40000 的 PE40, 它保留了完整的 ADP-核糖基化功能，但细胞毒性减低到 1%，这是由于 PE40 丧失细胞结合能力的结果。对 la区的定点诱变研究发现，当将第 57位赖氨酸（Lys57) 诱变为谷氨酸（Glu)时突变的 PE细胞毒性减低到 1%，这是由于突变毒素同靶细胞表面结合能力丧失的缘故。同时已观察到 Lys57位于 PE分子表面，这与其有受体结合能力是相符合的。 I区上其它 Lys位点的突变对 PE细胞毒素作用无任何影响。可见， Lys57是 la区的关键的氨基酸位点。除 Lys57夕卜，位于 la区的 His246， Arg247和 His249三个碱性氨基酸也是维持 PE生物学活性的重要位点。在突变型 PE的 Glu246， 247， 249和 PE Glu57, 246， 247， 249，上述碱性氨基酸已由 Glu替换，使 la区与 I I区间连接的氢键受阻断，突变型分子比天然 PE较为伸展，增加了对蛋白酶的敏感性，缩短了在小鼠体内循环的半衰期，因此，它们对动物显示低的细胞毒性。 The la region functions to recognize and bind to target cell surface receptors. The deletion of the la region produces a PE40 with a molecular weight of 40,000, which retains the intact ADP-ribosylation function, but the cytotoxicity is reduced to 1% due to the loss of PE40. The result of cell binding capacity. Site-directed mutagenesis studies on the la region revealed that the cytotoxicity of PE mutated when the 57th lysine (Lys57) was induced to glutamate (Glu) was reduced to 1% due to the mutation of the toxin with the target cell surface. The reason for the loss of binding ability. At the same time, it has been observed that Lys57 is located on the surface of PE molecules, which is consistent with its receptor binding ability. Mutations in other Lys sites on region I have no effect on PE cytotoxicity. It can be seen that Lys57 is a key amino acid site in the la region. In addition to Lys57, three basic amino acids, His246, Arg247 and His249, located in the la region, are also important sites for maintaining the biological activity of PE. In the mutant PE Glu246, 247, 249 and PE Glu57, 246, 247, 249, the above basic amino acid has been replaced by Glu, and the hydrogen bond linking the la region to the II region is blocked, and the mutant molecule is more than the natural PE. Stretching increases the sensitivity to proteases and shortens the half-life of circulation in mice, and therefore, they show low cytotoxicity to animals.

lb区在三维结构上位于 la区和 I I I区之间，此区的大部缺失并不影响 PE的生物学活性。在 PE40的基础上缺失了 lb区的第 365〜381位氨基酸，形成了 PE38分子。 PE38分子比 PE40分子量小 2000Da，缺失了 3_4个关键的抗原决定簇，因此抗原性明显降低。 The lb region is located between the la region and the I I I region in a three-dimensional structure, and most of the deletion in this region does not affect the biological activity of PE. On the basis of PE40, the amino acids 365 to 381 of the lb region were deleted to form PE38 molecules. The PE38 molecule has a molecular weight of 2000 Da less than PE40, and 3 to 4 key epitopes are deleted, so the antigenicity is significantly reduced.

I I区在毒素跨膜转位过程中起主导作用。当 I I区缺失时，尽管其细胞结合能力和 ADP-核糖基化活性尚存，但细胞毒性丧失，说明 I I区为毒素转位功能所必需的。 I I区的强疏水区***膜或横跨膜是转位过程的重要环节。 A与 B螺旋之间有一个环位于分子表面， Arg276和 Arg279在此环上，细胞蛋白酶裂解作用发生在第 279和第 280 位氨基酸之间。实验表明，将 Arg276、 Arg279点突变成 Gly后，其细胞毒性作用减低到 1/400。因为突变分子对细胞蛋白酶有抗力，因而不能裂解产生 37000片段，毒素不能转位入胞浆而不产生细胞毒作用。研究发现 Arg276不可用大小相近和电荷相同的氨基酸替换，说明此位点与细胞有关成分的相互作用是专一的。可见， Arg276 和 Arg279是维持 I I 区转位功能的关键性位点。其他重要的氨基酸位点有 Cys265， Cys287等，它们点突变成 Ser或 Ala后，造成细胞毒作用减低到 1/10。此外， I I区 E螺旋的部分（第 346〜364位氨基酸）缺失不引起 PE活性的丧失。 Zone II plays a leading role in the transmembrane translocation of toxins. When the region II is deleted, although its cell-binding ability and ADP-ribosylation activity persist, the cytotoxicity is lost, indicating that the region II is necessary for the toxin translocation function. Insertion of membrane or transmembrane into the membrane of the strong hydrophobic region of zone II is an important part of the translocation process. There is a ring between the A and B helices on the surface of the molecule, Arg276 and Arg279 are on this ring, and cell protease cleavage occurs between amino acids 279 and 280. Experiments have shown that after phasing Arg276 and Arg279 to Gly, the cytotoxicity is reduced to 1/400. Because the mutant molecule is resistant to cellular proteases, it cannot be cleaved to produce a 37000 fragment, and the toxin cannot be translocated into the cytosol without cytotoxicity. It was found that Arg276 could not be replaced by amino acids of similar size and charge, indicating that the interaction of this site with cell-related components is specific. It can be seen that Arg276 and Arg279 are key sites for maintaining the translocation function of region II. Other important amino acid sites are Cys265, Cys287, etc., which are mutated to Ser or Ala, resulting in a reduction in cytotoxicity to 1/10. In addition, the deletion of the portion of the E-helix of the region II (amino acids 346 to 364) did not cause loss of PE activity.

I I I区有三个功能，其一是催化 EF-2受 ADP-核糖基化作用，其二引导毒素的酶活性片段进入内质网。其三激活与细胞凋亡有关的 Caspases -3酶，启动细胞的凋亡机制而引起细胞凋亡。 Al lured提出 I I I区的一个裂缝区是酶活性中心。现已证实，定位在裂缝区的 Glu553是关键的活性位点， Glu553参与结合 NAD的过程， Glu553向 Asp 的点突变导致其 ADP-核糖基化活性至少减低到 1%，缺失它则完全丧失活性。其他几个裂缝区的残基 Arg458，Arg467及 Trp466也参与 I I I区同 NAD相互作用的过程。 Tyr481 以 Phe替代时引起 ADP-核糖基转移酶活性减低却不减低 NAD糖苷水解酶活性而被推测参与同 EF-2相互作用； Trp558也不直接参与结合 NAD的过程却为 ADP-核糖基化作用所必需。 I I I区的另一个关键残基 Hi s426位于由第 421〜432位氨基酸残基构成的 a螺旋内，此螺旋位于酶催化作用中心的远端，在 I I I区酶催化位点的分子构筑上起关键作用。此外， I I I区表面 Arg490附近是另一处蛋白酶靶区。 Arg490的缺失或突变可导致产生耐蛋白酶的分子， Arg492的缺失或突变引起 ADP-核糖基化活性减低。 I I I 区 C末端特异的氨基酸序列的功能是介导 PE 由胞吞泡向内质网的转位。此特异序列是 Arg609-Glu610-Asp61 1- Leu612_Lys613 ( S卩 REDLK)五个氨基酸残基片段。它的缺失使 PE的细胞毒性丧失，但对其 ADP-核糖基化活性并无任何影响。在活性 PE分子的 C末端特异序列 REDLK和使蛋白质在内质网滞留序列 KDEL之间有功能相似性。当编码 PE的 C末端 REDLK序列以 KDEL片段取代时可导致其细胞毒性提高 2_8 倍，证明 KDEL序列使 PE在内质网滞留作用更为有效。 The I I I region has three functions, one of which is to catalyze the ADP-ribosylation of EF-2, and the second to direct the enzymatically active fragment of the toxin into the endoplasmic reticulum. The three activate Caspases-3 enzymes involved in apoptosis, which initiate apoptosis of the cells and cause apoptosis. Al lured suggested that a fracture zone in the I I I region is the active center of the enzyme. It has been confirmed that Glu553 localized in the fracture region is a key active site, Glu553 is involved in the process of binding to NAD, and point mutation of Glu553 to Asp leads to at least 1% reduction of ADP-ribosylation activity, and complete loss of activity after deletion. . Residues Arg458, Arg467 and Trp466 in several other fracture regions are also involved in the interaction of I I I with NAD. When Tyr481 is substituted with Phe, it causes ADP-ribosyltransferase activity to decrease but does not decrease NAD glycoside hydrolase activity, and is presumably involved in interaction with EF-2; Trp558 is not directly involved in NAD-binding process but ADP-ribosylation Required. Another key residue of region III, Hi s426, is located in the a-helix consisting of amino acid residues 421 to 432, which is located at the distal end of the catalytic center of the enzyme, and plays a key role in the molecular construction of the catalytic site of the III region. effect. In addition, the surface of the I I I region near the Arg490 is another protease target region. Loss or mutation of Arg490 can result in the production of protease-resistant molecules, and deletion or mutation of Arg492 causes a decrease in ADP-ribosylation activity. The function of the C-terminal specific amino acid sequence of the I I I region is to mediate the translocation of PE from the endocytic vesicle to the endoplasmic reticulum. This specific sequence is a fragment of five amino acid residues of Arg609-Glu610-Asp61 1- Leu612_Lys613 (S卩 REDLK). Its deletion results in loss of cytotoxicity of PE, but has no effect on its ADP-ribosylation activity. There is a functional similarity between the C-terminal specific sequence REDLK of the active PE molecule and the protein endoplasmic reticulum retention sequence KDEL. When the C-terminal REDLK sequence encoding PE is substituted with the KDEL fragment, its cytotoxicity is increased by 2_8-fold, demonstrating that the KDEL sequence makes PE more effective in the retention of the endoplasmic reticulum.

用于构建本发明的融合蛋白质的 PE分子是在缺失了 la区和大部分 lb区的 PE 分子基础上，保留 609位 Arg和 611位 Asp , 将 Glu610、 Leu612、 Lys613分别突变为 Lys610、 Glu612和 Leu613，而形成一种新的氨基酸组合 RKDEL, 即 PE38m4a。 The PE molecule used to construct the fusion protein of the present invention is based on the deletion of the PE region of the la region and most of the lb region, retaining 609 Arg and 611 Asp, and mutating Glu610, Leu612, Lys613 to Lys610, Glu612 and Leu613, and formed a new amino acid combination RKDEL, namely PE38m4a.

鉴于本发明抗肿瘤嵌合毒素中导向剂或称识别分子的***释放激素的原序列基因不适合于基因工程表达，另外作为细胞毒性成分的 PE38m4a多位点突变，为了便于基因操作，人工设计合成了如下组分的基因序列： Ncol内切酶识别序列 -大肠杆菌偏性密码子组成的 mGnRH核苷酸序列，其中第 6位 Gly突变为 Trp-Ndel 内切酶识别序列 _PE38m4a核苷酸序列 -EcoRI内切酶识别序列。合成的基因序列的 5 ' 和 3 ' 端分别引入了 Nco l和 EcoRI核酸内切酶酶切位点，并造成适于连接的粘性末端。可按照本领域技术人员已知的重组技术（例如参见 Sambrook et al. , Mol ecular Cloning : A Laboratory Manual, Co ld Spring Harbor laboratory, 1989) , 克隆合成的基因入表达载体 pET27 (Merck Co. )系列中构建 pET27-mGnRH-PE38m4a。然后将重组载体***转化到原核细胞如大肠杆菌细胞中以生产 mGnRH-PE38m4a融合蛋白质。 DNA 重组操作中，使用限制性酶切法鉴定序列连接方向的正确性和可能的突变，最后以 Sanger双脱氧链终止法进行序列测定以进一步证实之。 In view of the fact that the original sequence gene of the gonadotropin-releasing hormone of the targeting agent or the recognition molecule of the anti-tumor chimeric toxin of the present invention is not suitable for genetic engineering expression, and the PE38m4a multi-site mutation as a cytotoxic component, for the convenience of gene manipulation, artificial The gene sequence of the following components was designed and synthesized: Ncol endonuclease recognition sequence - mGnRH nucleotide sequence consisting of E. coli bias codon, wherein the 6th Gly mutation is Trp-Ndel endonuclease The sequence _PE38m4a nucleotide sequence-EcoRI endonuclease recognition sequence was identified. The 5' and 3' ends of the synthetic gene sequence introduce Nco l and EcoRI endonuclease cleavage sites, respectively, and result in sticky ends suitable for ligation. The gene into the expression vector pET27 (Merck Co.) can be cloned according to recombinant techniques known to those skilled in the art (see, for example, Sambrook et al., Mol ecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, 1989). Construct pET27-mGnRH-PE38m4a. The recombinant vector system is then transformed into prokaryotic cells such as E. coli cells to produce the mGnRH-PE38m4a fusion protein. In the DNA recombination operation, restriction enzyme digestion was used to identify the correctness of the sequence joining direction and possible mutations, and finally the sequence was determined by Sanger dideoxy chain termination method to further confirm.

这里应特别指出的是，由于相对于细胞毒性部分 PE38m4a来说，导向部分 mGnRH 的分子量小得多，所以由两者产生的融合蛋白质很可能形成新的二级空间构象，导致 PE38m4a部分对 mGnRH的卷曲，影响融合蛋白质的受体结合的程度。然而通过技术人员计算机分析 PE38m4a部分的卷曲并不能影响 mGnRH的分子暴露和与受体结合。 It should be particularly noted here that since the molecular weight of the targeting moiety mGnRH is much smaller than that of the cytotoxic moiety PE38m4a, the fusion protein produced by both is likely to form a new secondary spatial conformation, resulting in a portion of PE38m4a to mGnRH. Curl, the extent to which receptor binding affects the fusion protein. However, analysis of the curl of the PE38m4a moiety by a technician computer does not affect the molecular exposure of mGnRH and binding to the receptor.

重组蛋白质基因将被可操纵地连接到适当的表达控制序列上，如连接到适于在大肠杆菌中使用的 T₇、 trp或 λ启动子、核糖体结合位点及转录终止信号上。可使用已知的转化方法，如适于原核细胞的氯化钙处理法将本发明的重组质粒转化到选择的宿主细胞中。可基于质粒上所含抗生素抗性基因所赋予的抗生素抗性来选择被转化的阳性细胞。一旦表达了所需的融合蛋白质，即可按照本领域已知的方法分离并纯化该融合蛋白质。例如，可从发酵培养物中离心收集菌体细胞并用溶菌酶和超声波裂解之，然后超速离心并在低浓度磷酸盐（约 20mM)溶液中加入饱和硫酸铵进行分步沉淀。依次经离子交换层析（IEC)和体积排阻层析（SEC)纯化所需的 mGnRH-PE38m4a 重组蛋白质。另外，亦可使用盐析、亲合层析及制备性凝胶电泳等方法进行纯化（参见 R. Scopes, Prote in Purif icaf ion, Springer-Verlag, N, Y. , 1982)。 The recombinant protein gene will be operably linked to appropriate expression control sequences, such as connecting to suitable for use in E. coli T _7, trp promoter or λ promoter, ribosome binding site and the transcription termination signal. The recombinant plasmid of the present invention can be transformed into a host cell of choice using known transformation methods, such as calcium chloride treatment for prokaryotic cells. The transformed positive cells can be selected based on the antibiotic resistance conferred by the antibiotic resistance gene contained on the plasmid. Once the desired fusion protein is expressed, the fusion protein can be isolated and purified according to methods known in the art. For example, the bacterial cells can be collected by centrifugation from the fermentation culture and lysed with lysozyme and ultrasonic waves, then ultracentrifuged and subjected to stepwise precipitation by adding saturated ammonium sulfate to a low concentration phosphate (about 20 mM) solution. The desired mGnRH-PE38m4a recombinant protein was purified by ion exchange chromatography (IEC) and size exclusion chromatography (SEC) in that order. Alternatively, it may be purified by methods such as salting out, affinity chromatography and preparative gel electrophoresis (see R. Scopes, Prote in Purif icafion, Springer-Verlag, N, Y., 1982).

一般说来，使用聚丙烯酰胺凝胶以 SDS-PAGE 电泳法（Laemml i, Nature 227 : 680-689, 1970)分析各柱层析洗脱部分，并使用多克隆抗 PE抗血清和以免疫印迹法检测之。对重组融合蛋白质纯化产物进行肿瘤细胞抑制试验以检测融合蛋白质的细胞毒性（IC₅。）。以及用 ¹²⁵1标记的天然 GnRH与融合蛋白在 A549细胞表面进行竞争取代，以确定融合蛋白中的 mGnRH与天然 GnRH对细胞表面受体的亲合能力。 In general, the elution fractions of each column were analyzed by SDS-PAGE electrophoresis (Laemml i, Nature 227: 680-689, 1970) using a polyacrylamide gel, and polyclonal anti-PE antiserum was used and immunoblotted. The law detects it. A tumor cell inhibition assay was performed on the recombinant fusion protein purified product to detect the cytotoxicity of the fusion protein (IC ₅ ). And the ¹²⁵ 1-labeled native GnRH and the fusion protein were competitively substituted on the surface of A549 cells to determine the affinity of mGnRH in the fusion protein to the native GnRH for cell surface receptors.

可将本发明的 mGnRH-PE38m4a 融合蛋白质作为基本活性成分，并加入一种或多种医药上可接受的载体或赋形剂，制成适于临床应用的药物组合物。所说的载体或赋形剂包括但不只限于磷酸盐缓冲盐水、生理盐水、等渗葡萄糖溶液、葡聚糖、右旋糖苷等。根据所治疗的疾病的不同，可在本发明的药物组合物中加入一种或多种与本发明的融合蛋白质有辅助或协同作用的其他天然的、合成的或重组的活性化合物。另外，可在本发明的药物组合物中加入选自人血清白蛋白、低分子量肽、甘氨酸或赖氨酸及金属阳离子（如 Zn²⁺、 Mn²⁺、 Mg²⁺和 Ca²⁺)的蛋白质保护剂，以及选自聚乙二醇、羧甲基纤维素、多聚甘氨酸、谷胱甘肽的稳定剂。 The mGnRH-PE38m4a fusion protein of the present invention can be used as a basic active ingredient, and one or more pharmaceutically acceptable carriers or excipients can be added to prepare a pharmaceutical composition suitable for clinical use. The carrier or excipient includes, but is not limited to, phosphate buffered saline, physiological saline, isotonic glucose solution, dextran, dextran, and the like. Depending on the disease being treated, one or more other natural, synthetic or recombinant active compounds which are auxiliary or synergistic with the fusion proteins of the invention may be added to the pharmaceutical compositions of the invention. In addition, a human serum albumin, a low molecular weight peptide, a glycine or a lysine, and a metal cation (such as Zn ²⁺ , Mn ²⁺ , Mg ^{2+ ,} and Ca ²⁺ ) may be added to the pharmaceutical composition of the present invention. a protein protectant, and selected from polyethylene glycol, carboxymethyl Stabilizer for cellulose, polyglycine, glutathione.

可以通过常规给药途径，特别是胃肠道外途径投用本发明的药物组合物，例如通过静脉内、腹腔内、肌肉内、皮内、皮下或粘膜内途径给药。本发明药物组合物的有效剂量范围可从几毫微克至几十毫克 /公斤体重 /天，但针对每个特定病人的具体用药剂量将根据待治疗疾病或病理状态的性质及严重程度、病人的年龄、体重、对药物的反应能力及给药方式等因素而定。 The pharmaceutical composition of the present invention can be administered by a conventional administration route, particularly a parenteral route, for example, by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous or intramucosal routes. The effective dosage of the pharmaceutical composition of the present invention may range from a few nanograms to several tens of milligrams per kilogram of body weight per day, but the specific dosage for each particular patient will depend on the nature and severity of the disease or pathological condition to be treated, the patient's Age, weight, ability to respond to drugs, and mode of administration.

应特别指出的是，尽管更深入的作用机理尚不明确，但我们的实验室已证明本发明的 mGnRH-PE38m4a蛋白质对包括结肠癌 HT-29细胞、卵巢癌 0VCAR3细胞、子宫颈腺癌 HeLa细胞及肝癌 HepG-2细胞等肿瘤细胞系均有明显的特异结合活性和细胞毒性；正常细胞基本没有致死作用；融合蛋白比天然 PEA活性提高了 9-10倍左右；而且成本低，方法简单，更适合工业化生产。附图说明 In particular, although the mechanism of action is not clear, our laboratory has demonstrated that the mGnRH-PE38m4a protein pair of the present invention includes colon cancer HT-29 cells, ovarian cancer 0VCAR3 cells, and cervical adenocarcinoma HeLa cells. And tumor cell lines such as HepG-2 cells have obvious specific binding activity and cytotoxicity; normal cells have almost no lethal effect; fusion protein is 9-10 times higher than natural PEA activity; and the cost is low, the method is simple, and more Suitable for industrial production. DRAWINGS

图 1显示用于表达 mGnRH-PE38m4a的重组质粒的构建图。 Figure 1 shows the construction of a recombinant plasmid for expression of mGnRH-PE38m4a.

图 2显示的 ¹²⁵I-mGnRH-PE38m4a (A)和 ¹²⁵I_GnRH (國）与 A549细胞膜表面受体结合的饱和曲线。 Figure 2 shows the saturation curves of ¹²⁵ I-mGnRH-PE38m4a (A) and ¹²⁵ I_GnRH (country) binding to A549 cell membrane surface receptors.

图 3显示的 ¹²⁵1标记的 mGnRH-PE38m4a和 GnRH与 A549细胞膜表面受体结合后分别用未标记的不同浓度的 GnRH或 mGnRH-PE38m4a竞争替代的曲线。 ♦代表不同浓度的 GnRH竞争替代 ¹²⁵I-mGnRH-PE38m4a的曲线，國代表不同浓度的 mGnRH_PE38m4a竞争替代 ¹²⁵I_GnRH的曲线。具体实施方式 Figure 3 shows a curve in which ¹²⁵ 1 -labeled mGnRH-PE38m4a and GnRH compete with A549 cell membrane surface receptors for competition with unlabeled different concentrations of GnRH or mGnRH-PE38m4a, respectively. ♦ Representing the different concentrations of GnRH to compete for the curve of ¹²⁵ I-mGnRH-PE38m4a, the country represents a different concentration of mGnRH_PE38m4a to compete for the curve of ¹²⁵ I_GnRH. detailed description

实施例 1 : mGnRH-PE38m4a融合蛋白质的制备 Example 1 : Preparation of mGnRH-PE38m4a fusion protein

A.重组表达质粒的构建与鉴定 A. Construction and identification of recombinant expression plasmid

a. mGnRH-PE38m4a双链基因的制备：参考现有文献资料设计并人工体外合成如 a. Preparation of mGnRH-PE38m4a double-stranded gene: refer to existing literature design and artificial in vitro synthesis

SEQ ID NO : 1所示 mGnRH-PE38m4a的双链核苷酸序列（含酶切识别位点及目的基因共 1089bp)。在 T₄连接酶（Promega)存在下直接克隆于 PGEM-T载体中，并转化大肠杆菌 JM105 , 获得含 PGEM-T/mGnRH-PE38m4a质粒的工程菌株。 The double-stranded nucleotide sequence of mGnRH-PE38m4a shown in SEQ ID NO: 1 (containing a restriction endonuclease recognition site and a gene of 1089 bp in total). In the T ₄ ligase (Promega) was cloned in a direct PGEM-T vector and transformed into E. coli JM105, obtaining engineered strains containing PGEM-T / mGnRH-PE38m4a plasmid.

b. mGnRH_PE38m4a表达质粒的制备：提取 PGEM_T/mGnRH_PE38m4a质粒，并利用 Ncol禾 P EcoRI核酸内切酶双酶切，收集 mGnRH_PE38m4a基因片段，在 T₄连接酶（Promega) 存在下直接克隆入相同酶切的 PET27 载体中，转化大肠杆菌 JM105 , 获得含 pET27-mGnRH-PE38m4a质粒的工程菌株。 Preparation of Expression Plasmid b mGnRH_PE38m4a: extracting PGEM_T / mGnRH_PE38m4a plasmid using Ncol cut Wo P EcoRI endonuclease digested, collected mGnRH_PE38m4a gene fragment was cloned directly into the same enzymes at T ₄ ligase (Promega) in the presence of In the PET27 vector, Escherichia coli JM105 was transformed to obtain an engineered strain containing the plasmid pET27-mGnRH-PE38m4a.

c mGnRH-PE38m4a表达菌株的筛选：提取 pET27-mGnRH_PE38m4a质粒进行酶切鉴定后，将正确克隆的质粒转化感受态大肠杆菌 BL21 ( A DE3)细胞，并将被转化的细胞培养于含卡那霉素（50 g/ml)的 LB培养基中，以扩增质粒 DNA。培养完成后，以常规方法提取质粒 DNA。用 Nco l和 EcoRI酶切鉴定，然后阳性重组质粒进行 DNA序列分析。 SEQ ID NO : 1显示了所测得的 mGnRH-PE38m4a重组基因的核苷酸序列。 SEQ ID N0 : 2显示了所测得的 mGnRH-PE38m4a重组基因的核苷酸序列所推导出的氨基酸序列。图 1显示了重组质粒 pET27-mGnRH-PE38m4a的构建。 Screening of c mGnRH-PE38m4a expression strain: After pET27-mGnRH_PE38m4a plasmid was extracted and identified by restriction enzyme digestion, the correctly cloned plasmid was transformed into competent E. coli BL21 (A DE3) cells, and the transformed cells were transformed. The cells were cultured in LB medium containing kanamycin (50 g/ml) to amplify the plasmid DNA. After the completion of the culture, the plasmid DNA was extracted in a conventional manner. Identification was carried out by digestion with Nco l and EcoRI, and then the positive recombinant plasmid was subjected to DNA sequence analysis. SEQ ID NO: 1 shows the nucleotide sequence of the measured mGnRH-PE38m4a recombinant gene. SEQ ID NO: 2 shows the amino acid sequence deduced from the nucleotide sequence of the measured mGnRH-PE38m4a recombinant gene. Figure 1 shows the construction of the recombinant plasmid pET27-mGnRH-PE38m4a.

B. mGnRH-PE38m4a融合蛋白质的表达及产物的纯化 B. Expression of mGnRH-PE38m4a fusion protein and purification of the product

将携带 pET27-mGnRH-PE38m4a重组基因质粒的大肠杆菌 BL21 ( λ DE3) (含有 T₇RNA 聚合酶启动子基因）（Studier, F. W. and Mof f att, B. A. , J. Mol, Biol , 189 : 113-130， 1986)培养在含有卡那霉素（50 μ g/ml)的 LB培养基中 37 °C培养，当 0D₆。。达到约 0. 4〜0. 6时加入 ImM异丙基硫代 _ β _D_半乳糖苷（IPTG) (终浓度 ImM)， 37 °C 继续培养 3-4小时，以诱导目的产物的表达。然后离心收集细菌，细菌经过适当方法破碎后再离心收集沉淀和上清，进行 SDS-PAGE 电泳确定目的蛋白的表达形式和表达量。经 SDS-PAGE 电泳表明目的蛋白以可溶性分泌形式表达，表达量占菌体总蛋白的 20%左右。并将含有目的蛋白质上清中加入缓冲液成分，终浓度达 50mM Tri s-HCl , pH8. 0, ImM EDTA, 4°C中空纤维超滤器（Mi l ipore)作用 30分钟， 4°C离心（20， 000g, 30分钟），取上清（可溶性部分）即为 mGnRH-PE38m4a粗提物。 E. coli BL21 (λ DE3) carrying the recombinant plasmid pET27-mGnRH-PE38m4a (containing the T ₇ RNA polymerase promoter gene) (Studier, FW and Mof f att, BA, J. Mol, Biol, 189: 113- 130, 1986) Culture in LB medium containing kanamycin (50 μg/ml) at 37 °C, when 0D ₆ . . When it was about 0. 4~0. 6, ImM isopropylthio-β_D_galactoside (IPTG) (final concentration ImM) was added, and incubation was continued at 37 °C for 3-4 hours to induce expression of the target product. Then, the bacteria are collected by centrifugation, and the bacteria are crushed by an appropriate method, and then the precipitate and the supernatant are collected by centrifugation, and the expression form and expression amount of the target protein are determined by SDS-PAGE electrophoresis. SDS-PAGE electrophoresis showed that the target protein was expressed in soluble secretion, and the expression amount was about 20% of total bacterial protein. The buffer containing the target protein supernatant was added to a buffer concentration of 50 mM Tri s-HCl, pH 8.0, ImM EDTA, 4 °C hollow fiber ultrafilter (Mi l ipore) for 30 minutes, and centrifuged at 4 ° C. (20, 000 g, 30 minutes), the supernatant (soluble fraction) is the crude extract of mGnRH-PE38m4a.

mGnRH-PE38m4a 粗提物经过缓冲液平衡的 DEAE-Sepharose Fast Flow 柱 (Pharmacia)，用含有 0〜0. 5M NaCl的 TE缓冲液（20mM Tri s-HCl, pH8. 0， ImM EDTA) 连续梯度洗脱，并收集蛋白质各组分峰部分。目的组分峰部分经小型中空纤维超滤器 (Mi l ipore)作用 30分钟超滤浓缩换液后，使浓缩物通过用 20mM Tri s-HCl， pH8. 0, ImM EDTA, 0. 15M NaCl 缓冲液平衡过的 1. 6 X 100cm Sephacryl-100 柱（Pharmacia) , 并用含有 0. 15M NaCl 的 TE缓冲液（20mM Tri s · HC1, pH8. 0， ImM EDTA)洗脱。收集活性峰部分并再次过高压液相色谱柱（日本岛津），收集蛋白质峰值 (A₂₈。）部分并在 30mM PBS 中彻底透析，透析后于 -20 °C下储存备用。如此纯化的蛋白质纯度 >95%。 The crude extract of mGnRH-PE38m4a was buffer-balanced on a DEAE-Sepharose Fast Flow column (Pharmacia), washed in a continuous gradient with TE buffer (20 mM Tri s-HCl, pH 8.0, ImM EDTA) containing 0 to 0.5 M NaCl. Remove and collect the peak fraction of each component of the protein. The peak of the target component was subjected to a small hollow fiber ultrafilter (Mi l ipore) for 30 minutes and concentrated by ultrafiltration. The concentrate was buffered by using 20 mM Tri s-HCl, pH 8.0, ImM EDTA, 0.15 M NaCl. The solution was equilibrated with a 1.6 X 100 cm Sephacryl-100 column (Pharmacia) and eluted with TE buffer (20 mM Tris. HC1, pH 8.0, ImM EDTA) containing 0.15 M NaCl. The active peak fraction was collected and again passed through a high pressure liquid chromatography column (Shimadzu, Japan), and the protein peak (A ₂₈ ) fraction was collected and thoroughly dialyzed in 30 mM PBS, and dialyzed and stored at -20 ° C until use. The purity of the protein thus purified is >95%.

利用 ¹²⁵1标记的天然 GnRH和 mGnRH_PE38m4a进行肿瘤细胞 A549的结合试验及多克隆抗 PE抗血清对纯化的 mGnRH-PE38m4a蛋白质进行鉴定。实施例 2 : ¹²⁵1标记的天然 GnRH和 mGnRH-PE38m4a融合蛋白质的靶特异性和生物学活性分析 The purified mGnRH-PE38m4a protein was identified by binding assay of tumor cell A549 with ¹²⁵ 1 labeled natural GnRH and mGnRH_PE38m4a and polyclonal anti-PE antiserum. Example 2: Target specificity and biological activity analysis of ¹²⁵ 1 labeled native GnRH and mGnRH-PE38m4a fusion proteins

A.结合能力试验 A. Binding ability test

a. GnRH禾口 mGnRH_PE38m4a的 ¹²⁵1标记：称取 1 mg Lodogen溶于 0. 5 ml氯仿中，取 50 μ 1 (100 μ ^加至试管底部，用氮气吹干，加不含保护剂的多肽或蛋白质半成品 0. 4ml , 加入 Na¹²⁵I 5mCi室温反应 12min，反应过程不断摇动，使之反应充分。 a. ¹²⁵ 1 label of GnRH and mGnRH_PE38m4a: Weigh 1 mg Lodogen dissolved in 0.5 ml of chloroform, take 50 μl (100 μ ^ to the bottom of the tube, blow dry with nitrogen, add the polypeptide without protective agent Or protein semi-finished product 0. 4ml, added Na ¹²⁵ I 5mCi reaction at room temperature for 12min, the reaction process is continuously shaken, so that the reaction is sufficient.

¹²⁵1标记的混合物利用 Sepharyl S-200 HR凝胶柱（1 X 50 cm)分离纯化，收集各管，取放射性强度最高的管进行试验。 ^{The 125} 1 labeled mixture was separated and purified using a Sepharyl S-200 HR gel column (1 X 50 cm). Tube, take the tube with the highest radioactivity for testing.

b.癌细胞及正常细胞培养：用 RPMI-1640完全培养基在 5%C0₂， 37 °C条件下单层贴壁培养并计数。每细胞孔中加入相同 CPM的 ¹²⁵1标记 GnRH、 mGnRH-PE38m4a (5 μ Ci/ 孔，稀释在 BSA-PBS中），并进行系列等倍稀释度， 1小时后 RPMI-1640完全培养基洗涤 5次，经胰蛋白酶消化，吹打收集细胞悬液，进行 Y计数（结果见图 2)。 b. Cancer cells and normal cell culture: Monolayer adherent cultures were counted and counted in RPMI-1640 complete medium at 5% CO ₂ , 37 °C. Add ¹²⁵ 1 labeled GnRH, mGnRH-PE38m4a (5 μ Ci/well, diluted in BSA-PBS) of the same CPM to each well, and perform serial dilutions of the series. After 1 hour, RPMI-1640 complete medium wash 5 The cells were collected by trypsinization, pipetting, and counted by Y (see Figure 2 for results).

从图 2所示的结果可以看出， ¹²⁵1标记 GnRH和 mGnRH_PE38m4a均以剂量依赖方式结合到肿瘤细胞 A549表面直到达到饱和程度，并且显示 ¹²⁵I-mGnRH-PE38m4a的结合能力〉 ¹²⁵I-GnRH，而对正常细胞结合能力均极低，从而证实本发明部分中纯化的 mGnRH-PE38m4a融合蛋白能够以剂量依赖方式与受体阳性细胞结合。 As can be seen from the results shown in Fig. 2, both ¹²⁵ 1-labeled GnRH and mGnRH_PE38m4a bind to the surface of tumor cell A549 in a dose-dependent manner until saturation is reached, and the binding ability of ¹²⁵ I-mGnRH-PE38m4a is shown to be > ¹²⁵ I-GnRH, The ability to bind to normal cells was extremely low, thus confirming that the purified mGnRH-PE38m4a fusion protein in the present invention can bind to receptor-positive cells in a dose-dependent manner.

B. GnRH、 mGnRH_PE38m4a与 ¹²⁵1标记 GnRH、 mGnRH_PE38m4a竞争结合试验基本上按照 Qayum, A.等人（Br. J. Cancer 62： 96-99， 1990)所述的方法进行多肽或蛋白质对 A549细胞结合的特异性竞争和取代研究。 B. GnRH, mGnRH_PE38m4a and ¹²⁵ 1 labeled GnRH, mGnRH_PE38m4a competition binding assay The peptide or protein binding to A549 cells was performed essentially as described by Qayum, A. et al. (Br. J. Cancer 62: 96-99, 1990). Specific competition and substitution studies.

将培养的 A549单层细胞记数后加入结合试验饱和曲线拐点处放射性标记物 Y计数单位的 ¹²⁵I_mGnRH并 37 °C温浴 1小时。 RPMI-1640完全培养基洗涤 5次， 1小时后再分别加入未标记的 mGnRH-PE38m4a (0. 01-1000 μ M)， 37 °C温浴 1 小时， RPMI-1640 完全培养基洗涤 5次，经胰蛋白酶消化，吹打收集细胞悬液，进行 Y计数；另外进行反证试验：将培养的 A549单层细胞计数后加入饱和曲线拐点处放射性标记物 Y计数单位的 ¹²⁵I-mGnRH-PE38m4a，用未标记的 GnRH (0. 01-1000 μ M)进行竞争和取代研究，结果见图 3。 The cultured A549 monolayer cells were counted and added to ¹²⁵ I_mGnRH of the radioactive marker Y counting unit at the inflection point of the test saturation curve and incubated at 37 ° C for 1 hour. Wash RPMI-1640 complete medium 5 times, add unlabeled mGnRH-PE38m4a (0. 01-1000 μ M) after 1 hour, warm bath at 37 °C for 1 hour, wash RPMI-1640 complete medium for 5 times, Trypsin digestion, pipetting to collect cell suspension, and performing Y counting; additionally performing a reverse test: the cultured A549 monolayer cells were counted and added to the ¹²⁵ I-mGnRH-PE38m4a of the radioactive marker Y counting unit at the inflection point of the saturation curve, with unlabeled The GnRH (0. 01-1000 μ M) was subjected to competition and substitution studies, and the results are shown in Figure 3.

图 3中國代表不同浓度的 mGnRH-PE38m4a竞争替代 ¹²⁵I_GnRH的曲线。 ♦代表不同浓度的 GnRH竞争替代 ¹²⁵I-mGnRH-PE38m4a的曲线。 Figure 3 shows a curve in which China's different concentrations of mGnRH-PE38m4a compete for ¹²⁵ I_GnRH. ♦ Representing a different concentration of GnRH to compete for the curve of ¹²⁵ I-mGnRH-PE38m4a.

从图 3所示的数据可以看出，以渐增的浓度加入未标记的 mGnRH-PE38m4a或 GnRH 均可以将标记物进行取代，另外可以看出 GnRH 取代 ¹²⁵I-mGnRH-PE38m4a 的浓度是 mGnRH-PE38m4a取代 ¹²⁵I_GnRH的浓度的 7_8倍左右，说明 mGnRH_PE38m4a融合蛋白中经过突变处理，其中的 mGnRH组分较未突变的 GnRH对靶细胞的结合能力提高 7倍以上。实施例 3 : 细胞毒性试验 It can be seen from the data shown in Fig. 3 that the label can be substituted by adding unlabeled mGnRH-PE38m4a or GnRH at an increasing concentration, and it can be seen that the concentration of GnRH substituted ¹²⁵ I-mGnRH-PE38m4a is mGnRH- PE38m4a replaced about 7-8 times the concentration of ¹²⁵ I_GnRH, indicating that the mGnRH_PE38m4a fusion protein was subjected to mutation treatment, and the mGnRH component was more than 7-fold more binding to the target cell than the unmutated GnRH. Example 3: Cytotoxicity test

定量的样品经过滤除菌，按等倍稀释法将不同量的样品加入各细胞孔中，使之总体积为 100 μ 1， 5%C0₂， 37 °C条件下培养 12h，培养板各孔中分别加入 100 μ 1 ΜΤΤ染色试剂， 5%C0₂， 37 °C条件下继续培养 4h，于 490nm波长下测定吸光值，计算重组毒素对各种肿瘤和正常细胞的 50%细胞死亡的浓度（IC₅。），同时利用天然绿脓杆菌外毒素 A (PEA, Sigma公司）作为对照，比较融合毒素和天然 PEA的活性变化。结果见表 1。表 1 : 纯化的 mGnRH-PE38m4a和天然 PEA对肿瘤和正常细胞的 IC: Quantitative samples were filtered and sterilized. Different amounts of samples were added to each cell well by equal dilution method, and the total volume was 100 μl, 5% C0 ₂ , and cultured at 37 °C for 12 h. Add 100 μl ΜΤΤ staining reagent, 5% C0 ₂ , 37 °C for 4 h, measure the absorbance at 490 nm, and calculate the concentration of recombinant toxin on 50% cell death of various tumors and normal cells ( IC ₅ .), while using Pseudomonas aeruginosa exotoxin A (PEA, Sigma) as a control, compares the changes in activity of the fusion toxin and natural PEA. The results are shown in Table 1. Table 1: ICs of purified mGnRH-PE38m4a and native PEA for tumor and normal cells:

表 1显示纯化的 mGnRH-PE38m4a和天然 PEA对某些肿瘤细胞及某些正常细胞培养物的细胞毒性。 IC₅。数值是指杀死靶细胞达到对照组的 50%所需的融合蛋白的浓度。 A549 为人肺腺癌细胞株、 L0V0为人肠癌细胞株、 MKN45 为人胃腺癌细胞株、 Bcap37 为人乳腺癌细胞株、 QGY为人肝癌细胞株、 PC-3M为人***癌细胞株、 A375为人黑色素瘤细胞株、 KB为人口腔癌细胞株，正常细胞有人胚肾、人胚肝培养株。

Table 1 shows the cytotoxicity of purified mGnRH-PE38m4a and native PEA on certain tumor cells and certain normal cell cultures. IC ₅ . The value refers to the concentration of the fusion protein required to kill the target cells to 50% of the control group. A549 is a human lung adenocarcinoma cell line, L0V0 is a human intestinal cancer cell line, MKN45 is a human gastric adenocarcinoma cell line, Bcap37 is a human breast cancer cell line, QGY is a human liver cancer cell line, PC-3M is a human prostate cancer cell line, and A375 is a human melanoma cell line. KB is a human oral cancer cell line, and normal cells are human embryo kidney and human embryo liver culture strain.

从表 1所示的结果可以看出，本发明所纯化的 mGnRH-PE38m4a可以杀死人肺腺癌、人肠癌、人胃腺癌、人乳腺癌、人肝癌、人***癌、人黑色素瘤、人口腔癌细胞株，并且 IC₅。值较低，而对正常细胞人胚肾、人胚肝培养株无杀伤作用；天然 PEA 对相同种类肿瘤细胞的 IC₅。均高于融合蛋白 IC₅。值，二者相差 10倍左右，由此可见，融合蛋白比天然 PEA活性提高了 9-10倍左右。试验还表明 PEA几乎对所有细胞都有致死作用，而 mGnRH-PE38m4a则对正常细胞基本没有致死作用。 As can be seen from the results shown in Table 1, the purified mGnRH-PE38m4a of the present invention can kill human lung adenocarcinoma, human intestinal cancer, human gastric adenocarcinoma, human breast cancer, human liver cancer, human prostate cancer, human melanoma, Human oral cancer cell line, and IC ₅ . The value is lower, and has no killing effect on normal cell human embryonic kidney and human embryo liver culture strain; natural PEA has IC ₅ on the same kind of tumor cells. Both are higher than the fusion protein IC ₅ . The value, the difference between the two is about 10 times, which shows that the fusion protein is 9-10 times more effective than the natural PEA activity. The test also showed that PEA has a lethal effect on almost all cells, while mGnRH-PE38m4a has no lethal effect on normal cells.

Claims

权利要求 Rights request

1、靶特异性双突变体融合蛋白质，它是由突变的***释放激素 mGnRH与重组绿脓杆菌外毒素 A突变体融合而成的融合毒素，其特征在于所述的重组绿脓杆菌外毒素是去除 la区及 lb区的氨基酸 365-380，且 C末端氨基酸 Glu610、Leu612、Lys613 分别人工突变为 Lys610、 Glu612和 Leu613的 PEA分子，即 PE38m4a。 1. A target-specific double mutant fusion protein, which is a fusion toxin fused by a mutant gonadotropin releasing hormone mGnRH and a recombinant Pseudomonas aeruginosa exotoxin A mutant, characterized by the recombinant Pseudomonas aeruginosa The toxin is the amino acid 365-380 which removes the la and lb regions, and the C-terminal amino acids Glu610, Leu612, and Lys613 are artificially mutated to the PEA molecules of Lys610, Glu612 and Leu613, respectively, ie, PE38m4a.

2、根据权利要求 1所述的靶特异性双突变体融合蛋白质，其特征在于：所述的突变的***释放激素 mGnRH 的基因是以大肠杆菌偏性密码子为主体的基因形式合成的，氨基酸序列为： Met Gly Glu His Trp Ser Tyr Trp Leu Arg Pro Gly His。 2. The target-specific double mutant fusion protein according to claim 1, wherein: the gene of the mutated gonadotropin releasing hormone mGnRH is synthesized by a gene form mainly composed of an Escherichia coli bias codon. The amino acid sequence is: Met Gly Glu His Trp Ser Tyr Trp Leu Arg Pro Gly His.

3、靶特异性双突变体融合蛋白质，它的基因序列如序列表 SEQ ID NO : 1所示。 3. A target-specific double mutant fusion protein, the gene sequence of which is shown in SEQ ID NO: 1 of the sequence listing.

4、靶特异性双突变体融合蛋白质，它的氨基酸序列如序列表 SEQ ID NO : 2 所示。 4. A target-specific double mutant fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 2 of the Sequence Listing.

5、一种表达载体， pET27-mGnRH-PE38m4a，其基因序列中含有序列表 SEQ ID NO : 1所示的序列。 An expression vector, pET27-mGnRH-PE38m4a, comprising the sequence shown in SEQ ID NO: 1 in the gene sequence.

6、靶特异性双突变体融合蛋白质，是由下述方法制备的： 6. A target-specific double mutant fusion protein, which is prepared by the following method:

(4)在适当条件下表达所说的融合蛋白质； (4) expressing said fusion protein under appropriate conditions;

(5)从细胞培养物中回收并纯化融合蛋白质。 (5) The fusion protein is recovered and purified from the cell culture.

7、根据权利要求 6所述的靶特异性双突变体融合蛋白质，其特征在于：步骤 (1) 所述的 mGnRH基因序列，采用人工设计合成了 Ncol 内切酶识别序列一大肠杆菌偏性密码子组成的 mGnRH核苷酸序列，其中第 6位 Gly突变为 Trp—Ndel内切酶识别序列 —PE38m4a核苷酸序列一EcoRI内切酶识别序列；合成的基因序列的 5 ' 和 3 ' 端分别引入了 Ncol和 EcoRI核酸内切酶酶切位点，并造成适于连接的粘性末端。 The target-specific double mutant fusion protein according to claim 6, wherein: the mGnRH gene sequence of step (1) is artificially designed to synthesize an Ncol endonuclease recognition sequence-E. coli bias code. a mGnRH nucleotide sequence consisting of a 6th Gly mutation to a Trp-Ndel endonuclease recognition sequence-PE38m4a nucleotide sequence-EcoRI endonuclease recognition sequence; the 5' and 3' ends of the synthesized gene sequence are respectively Ncol and EcoRI endonuclease cleavage sites were introduced and resulted in sticky ends suitable for ligation.

8、根据权利要求 7所述的靶特异性双突变体融合蛋白质，其特征在于：步骤 (2) 所述的基因如序列表 SEQ ID NO : 1所示。 The target-specific double mutant fusion protein according to claim 7, wherein the gene of the step (2) is as shown in SEQ ID NO: 1.

9、根据权利要求 6、 7、 8所述的靶特异性双突变体融合蛋白质，其特征在于：所述的载体是 PET27，步骤 (3)所述的大肠杆菌为大肠杆菌 BL21 ( λ DE3)。 The target-specific double mutant fusion protein according to claims 6, 7, or 8, wherein the carrier is PET27, and the Escherichia coli according to the step (3) is Escherichia coli BL21 (λ DE3). .

10、根据权利要求 1、 2、 3、 4、 6、 7、 8、 9所述的靶特异性双突变体融合蛋白质，在治疗多种与***释放激素受体有关的肿瘤药物的应用。 10. The target-specific double mutant fusion protein according to claims 1, 2, 3, 4, 6, 7, 8, 9 for use in the treatment of a plurality of tumor drugs associated with gonadotropin releasing hormone receptors .