【作者】 刘艳华；

【导师】 陈薇； 王景林；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2008， 博士

【摘要】 重组免疫毒素是由靶向区和细胞毒性区组成、可以识别并选择性杀伤特定细胞的融合蛋白,是一类重要的肿瘤靶向性治疗药物。靶向区为能与肿瘤细胞表面抗原特异结合的抗体、细胞因子或激素,毒性区为细菌毒素,如铜绿假单胞菌外毒素(PE)和白喉毒素(DT),或植物毒素,如蓖麻毒素(RT)、美洲商陆抗病毒蛋白(PAP)和皂草素等。重组免疫毒素用于恶性肿瘤的导向治疗已取得很大成功,如以人IL-2为导向分子,以DT为毒性部分的免疫毒素制剂(商品名ONTKA)已获得美国FDA批准,用于治疗皮肤T细胞淋巴瘤(CTCL),另有多种重组免疫毒素在临床试验研究中也显示出良好的抗肿瘤效果。尽管重组免疫毒素具有广阔的应用前景,但是在临床应用中仍面临许多挑战,如免疫原性强和穿透性差,解决方法之一就是构建小分子的重组免疫毒素。丝瓜毒素(luffin)LuffinP1和luffinS2是从丝瓜籽中分离到的两种只有5kDa和8kDa的小分子量I型核糖体失活蛋白(RIP),具有N-糖苷酶活性,能特异性水解真核细胞核糖体28s rRNA 4324位的腺嘌呤,使真核细胞核糖体60s大亚基失活,从而抑制蛋白质合成,对兔网织红细胞裂解液的IC50分别为10-9和10-10M左右,可作为潜在的免疫毒素弹头分子。人Ⅰ型促性腺激素释放激素( gonadotropin-releasing hormone, GnRH或luteinizing hormone-releasing hormone, LHRH)是下丘脑分泌的一种十肽类激素,用于调控脑垂体促黄体生成素(LH)和促卵泡激素(FSH)的分泌。GnRH受体通常分布于脑垂体、性腺的细胞膜上,但研究发现,GnRH受体也高表达于某些癌细胞表面,如乳腺癌、卵巢癌、子宫内膜癌、胰腺癌、前列腺癌及肝癌细胞等。因此GnRH和相关肽已经应用于妇科学和肿瘤学领域的研究,如用于性腺机能减退、不育症和性腺依赖性肿瘤的治疗。另外,肿瘤细胞表面的GnRH受体与GnRH有较高的亲和力,因此GnRH可以作为肿瘤靶向治疗中的导向分子。如制备的LHRH-PE40重组免疫毒素对人多种肿瘤细胞系有明显的杀伤作用,目前已进入临床试验阶段。因此,以GnRH为导向分子、以luffinP1或luffinS2为毒性部分构建的重组免疫毒素是一种潜在的免疫原性低、穿透力强的小分子抗肿瘤药物。本文首先克隆并原核表达了luffinP1和luffinS2蛋白,测定其蛋白合成抑制作用,筛选活性较高的蛋白作为重组免疫毒素的毒性部分;随后将GnRH与luffinS2进行基因融合,原核表达并纯化GnRH-luffinS2和luffinS2-GnRH融合毒素,测定其体外细胞毒作用;因为铜绿假单胞菌外毒素转膜区(PEⅡ区)具有蛋白酶识别位点和转膜功能,能提高重组免疫毒素的转膜效率,并能使重组免疫毒素的毒性部分和导向部分在胞内分离,从而提高重组免疫毒素的细胞毒作用,所以将其插入到GnRH和luffinS2之间,构建GnRH-PEⅡ-luffinS重组免疫毒素,表达纯化后测定其体内外抑肿瘤活性。具体的实验内容和结果如下:1. luffinP1和luffinS2全长基因的克隆、表达和活性研究(1)利用Codon软件,设计luffinP1和luffinS2的重叠PCR引物,通过对重叠PCR反应体系和条件的优化,扩增得到了luffinP1和luffinS2的全长基因。(2)luffinP1和luffinS2分别与表达载体pET-His、pET32a、pQE30和pBV220连接,构建pET-His/luffinP1、pET-His/luffinS2、pET32a/luffinP1、pET32a/luffinS2、pQE30/luffinP1、pQE30/luffinS2、pBV220/luffinP1和pBV220/luffinS2表达载体,转化宿主菌,并诱导表达。结果只有pET32a/luffinP1和pET32a/luffinS2转化BL21(DE3)大肠埃希杆菌后获得表达,且均为可溶性表达。经亲和层析纯化后,获得Trx-luffinP1和Trx-luffinS2融合蛋白。随后,利用重组肠激酶(rEK)将Trx-luffinP1和Trx-luffinS2融合蛋白切割,获得rluffinP1和rluffinS2。(3)测定rluffinP1和rluffinS2对兔网织红细胞裂解液的蛋白合成抑制作用。结果表明,rluffinP1和rluffinS2的IC50分别为9.51μg/ml和1.58μg/ml,因此选择luffinS2作为重组免疫毒素的毒性部分。2. GnRH-luffinS2和luffinS2-GnRH融合蛋白的克隆、表达和活性研究(1)因为无法确定GnRH和luffinS2的连接顺序对GnRH和luffinS2的活性是否有影响,因此设计了GnRH-luffinS2(GnRH在N端)和luffinS2-GnRH(GnRH在C端)融合蛋白,并对其二级进行预测。(2)利用Codon软件,设计了GnRH-luffinS2和luffinS2-GnRH的重叠PCR引物,通过对重叠PCR反应体系和条件的优化,扩增得到了GnRH-luffinS2和luffinS2-GnRH的全长基因。(3)GnRH-luffinS2和luffinS2-GnRH分别与pET-His、pET32a、pQE30和pBV220连接,构建pET-His/GnRH-luffinS2、pET-His/luffinS2-GnRH、pET32a/GnRH-luffinS2、pET32a/luffinS2-GnRH、pQE30/GnRH-luffinS2、pQE30/luffinS2-GnRH、pBV220/GnRH-luffinS2和pBV220/luffinS2-GnRH原核表达载体,转化宿主菌,并诱导表达。结果只有pET32a/GnRH-luffinS2和pET32a/luffinS2-GnRH转化BL21(DE3)后获得表达,且均为可溶性表达。经亲和层析纯化后,获得了Trx-GnRH-luffinS2和Trx-luffinS2-GnRH。随后,利用rEK将Trx-GnRH-luffinS2和Trx-luffinS2-GnRH融合蛋白切割,获得rGnRH-luffinS2和rluffinS2-GnRH。(4)采用XTT法测定rGnRH-luffinS2和rluffinS2-GnRH对体外肿瘤细胞Hela、A549、HepG-2、SP2/0和CEF细胞毒性作用。结果表明,rGnRH-luffinS2对上述细胞的IC50分别为67.19μg/ml、70.42μg/ml、84.44μg/ml和106.25μg/ml,rluffinS2-GnRH对上述细胞的IC50分别79.5μg/ml、76.7μg/ml、96.2μg/ml和137μg/ml,两种融合毒素对CEF均无毒性,表明rGnRH-luffinS2和rluffinS2-GnRH具有一定的抗肿瘤作用,但活性不高。3. GnRH-PEⅡ-luffinS2融合蛋白的克隆、表达和抗肿瘤活性研究( 1 )利用PEⅡ具有转膜区和蛋白酶识别位点的特性,设计GnRH-PEⅡ-luffinS重组毒素并对其二级进行预测。(2)利用Codon软件,设计了GnRH-PEⅡ-luffinS的重叠PCR引物,通过对重叠PCR反应体系和条件的优化,扩增得到了GnRH-PEⅡ-luffinS的全长基因。(3)选择pET32a为表达载体,构建pET32a/GnRH-PEⅡ-luffinS表达载体,转化BL21(DE3)大肠埃希杆菌后获得表达,蛋白以包涵体形式存在。经亲和层析纯化后,获得了Trx-GnRH-PEⅡ-luffinS融合蛋白,蛋白经透析复性和超滤浓缩后,利用rEK将Trx-GnRH-PEⅡ-luffinS融合蛋白切割为Trx和rGnRH-PEⅡ-luffinS。(4)采用XTT法测定rGnRH-PEⅡ-luffinS在体外对Hela、A549、HepG-2、SP2/0和CEF细胞毒性作用。结果表明,rGnRH-PEⅡ-luffinS对上述细胞的IC50分别为13.50μg/ml、13.74μg/ml、16.79μg/ml和26.07μg/ml,对CEF无作用,表明具有较强的抗肿瘤作用。(5)建立BALB/c小鼠的SP2/0实体瘤模型,检测rGnRH-PEⅡ-luffinS体内抗肿瘤活性。结果表明,剂量为100μg/只,瘤区内穿刺注射,连续10d给药时,rGnRH-PEⅡ-luffinS对SP2/0实体瘤的抑瘤率为50.3%,显示rGnRH-PEⅡ-luffinS具有一定的抗肿瘤作用。综上所述,本研究原核表达了luffinP1和luffinS2蛋白,利用兔网织红细胞裂解液证实luffinS2具有较高的蛋白合成抑制作用;然后构建了以GnRH为导向分子,以luffinS2为毒性分子的小分子重组免疫毒素GnRH-luffinS2和luffinS2-GnRH,体外对Hela、A549、HepG-2、SP2/0细胞毒实验表明,GnRH-luffinS2和luffinS2-GnRH的细胞毒性较低;为提高重组免疫毒素的细胞毒性,将PEⅡ插入到GnRH和luffinS之间,构建了GnRH-PEⅡ-luffinS重组免疫毒素。体外细胞毒实验表明,其细胞毒性获得明显提高,体内抑瘤实验表明,GnRH-PEⅡ-luffinS具有一定抗肿瘤活性,为进一步探讨其作为抗肿瘤药物的临床应用奠定了基础。更多还原

【Abstract】 Recombinant immunotoxin is an important drug of targeted cancer therapy and is composed of a very potent protein toxin and a targeting moiety such as a recombinant antibody fragment or growth factor. Protein toxins commonly used include bacterial toxins such as PE and DT, and plant toxins such as ricin, PAP and saporin. The clinical efficacy of recombinant immunotoxins has been demonstrated in patients with malignant tumors. For example, recombinant immunotoxin ONTAK, which consists of truncated DT and IL2, has been approved by the FDA for CTCL therapy. But the antitumor activity of recombinant immunotoxins in vivo is interfered by many factors, e.g, good immunogenicity and poor penetration into tumors. One of methods is to develop new type of micromolecule recombinant immunotoxins to reduce their immunogenicity and improve their penetration into tumor cells.LuffinP1 and luffinS2, seperated from the seeds of Luffa cylindrical, are two novel ribosome inactivating proteins (RIPs).The two small toxin molecules are RNA N-glycosidases with molecular weights of 5 kDa and 8 kDa, respectively, and are much smaller than any other RIPs so far investigated. Their IC50 values are 10-9M and 10-10M respectively and can be used as cytotoxic moiety of recombinant immunotoxins. TypeⅠof the gonadotropin releasing-hormone (GnRH), also called luteinizing hormone-releasing hormone (LHRH), is a neuro-decapeptide. After binding to its receptor on pituitary cells, it triggers the secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH). These gonadotropin hormones control the activity of the gonads. GnRH receptors have been identified on tumor cells from mammary, endometrial, prostatic, hepatic and pancreatic tissues as well as on tumor cell lines. A growing interest has developed for the application of GnRH and related peptides in the fields of gynaecology and oncology. GnRH and its analogues have been used in treatment of hypogonadism and infertility. It is also used for the treatment of gonadotropin dependent cancers. Furthermore, this peptide binds to its receptor with high affinity (kd about 10-9M) and is not immunogenic, so GnRH is a very good candidate for the targeted moiety of recombinant immunotoxins. For example, LHRH-PE40 showed antitumor efficacy in preclinical study and has been produced for clinical phase testing. Therefore, recombinant immunotoxins, composed of GnRH used as targeted moiety and luffinP1 or luffinS2 used as cytotoxic moiety, are potential antitumor drugs which have low molecular weights, poor immunogenicity and strong penetration.We firstly cloned and expressed luffinP1 and luffinS2 in E. coli, and analyzed their protein biosynthesis inhibitory activity in cell-free systems. The protein with higher activity was used to construct recombinant immunotoxins along with GnRH. Then, the two recombinant immunotoxins of GnRH-luffinS2 and luffinS2-GnRH were designed and constructed. After the fusion proteins were expressed and purified, the in vitro cytotoxicity of the fusion proteins was determined by XTT method. Finally, because PEⅡfragment has the function of translocation and a protease recognition site, which can enhance efficacy of transmembrane and make targeted moiety and toxic moiety separate in cytoplasm, we constructed a novel recombinant immunotoxin GnRH-PEⅡ-luffinS, in which PEⅡw as inserted into GnRH-luffinS2, and analyzed the antitumor activity of the fusion protein in vitro and in vivo.In this study, the experiments were conducted as follow.1. Cloning, expression and activity assay of luffinP1 and luffinS2(1) The PCR primers were designed by Condon software, after PCR conditions were optimized, the genes of luffinP1 and luffinS2 were amplified. (2) LuffinP1 and luffinS2 were ligated with expression vectors pET-His, pET32a, pQE30 and pBV220, and recombinant vectors pET-His/luffinP1, pET-His/luffinS2, pET32a/luffinS2, pET32a/luffinP1, pQE30/luffinP1, pQE30/luffinS2, pBV220/luffinP1 and pBV220/luffinS2 were constructed. The results of expression showed that only recombinant vectors pET32a/luffinS2 and pET32a/luffinP1 of them can express target fusion proteins in soluble form. The soluble fusion proteins were purified by Ni-NTA chromatography method. After cleaved by rEK, the single rluffinP1 and rluffinS2 were produced. (3) Then, the protein bioinhibitory activitives were determined by rabbit reticulocyte lysate. The results showed that IC50 of rluffinP1 and rluffinS2 were 9.51μg/ml and 1.58μg/ml, respectively. So luffinS2 was selected as the cytotoxic moiety of recombinant immunotoxins in following experiment.2. Design, cloning, expression and activity assay of GnRH-luffinS2 and luffinS2-GnRH(1) Because the order of GnRH and luffinS2 may influence their activitives, the GnRH-luffinS2, which GnRH was fused to the N terminus of luffinS2, and the luffinS2-GnRH, which GnRH was fused to the C terminus of luffinS2, were designed, and their secondary structures were anticipated. (2) The PCR primers were designed by Condon software, the gene fragments of GnRH-luffinS2 and luffinS2-GnRH were amplified by optimized PCR reaction conditions. (3) the two gene fragments of GnRH-luffinS2 and luffinS2-GnRH were respectively ligated with expression vectors pET-His, pET32a, pQE30 and pBV220, and recombinant vectors pET-His/GnRH-luffinS2, pET-His/luffinS2-GnRH, pET32a/GnRH-luffinS2, pET32a/luffinS2-GnRH, pQE30/GnRH-luffinS2, pQE30/luffinS2-GnRH, pBV220/GnRH-luffinS2 and pBV220/luffinS2-GnRH were all constructed. The results of expression showed that only recombinant vectors of pET32a/GnRH-luffinS2 and pET32a/luffinS2-GnRH can express target fusion proteins in soluble form. The soluble fusion proteins were purified by Ni-NTA chromatography method. After cleaved by rEK, the rGnRH-luffinS2 and rluffinS2-GnRH were produced. (4) Then their cytotoxicity to Hela, A549, HepG-2, SP2/0 and CEF cells were analyzed by XTT method. The results showed that IC50 values of rGnRH-luffinS2 were 67.19μg/ml for Hela cells, 70.42μg/ml for A549 cells, 84.44μg/ml for HepG-2 cells and 106.25μg/ml for SP2/0 cells, respectively. The IC50 values of rluffinS2-GnRH were 79.5μg/ml for Hela cells, 76.7μg/ml for A549 cells, 96.2μg/ml for HepG-2 cells and 137μg/ml for SP2/0 cells, respectively. But the two fusion proteins showed no toxic effect to CEF cells. The results revealed that GnRH-luffinS2 and luffinS2-GnRH had low cytotoxicity to cancer cell lines3. Design, cloning, expression and activity assay of GnRH-PEⅡ-luffinS(1) Because PEⅡhas the function of translocation and includes a protease recognition site, recombinant immunotoxin GnRH-PEⅡ-luffinS was designed, in which PEⅡwas inserted into GnRH-luffinS2, and its secondary structure was anticipated. (2) The PCR primers were designed by Condon software, the gene fragment of GnRH-PEⅡ-luffinS was amplified by optimized PCR reaction conditions. (3) GnRH-PEⅡ-luffinS gene was inserted into expression vector pET32a and transformed into E.coli BL21. The recombinant plasmid pET32a/GnRH-PEⅡ-luffinS expressed insoluble protein. The inclusion bodies of fusion protein were extracted and purified by Ni-NTA chromatography method. After renaturation through dialysis and concentration through ultrafiltration, the fusion protein was cleaved by rEK in order to produce rGnRH-PEⅡ-luffinS. (4) The cytotoxicity to Hela, A549, HepG-2, SP2/0 and CEF cells was analyzed by XTT method. The result showed that IC50 values of rGnRH-PEⅡ-luffinS were 13.50μg/ml for Hela cells, 13.74μg/ml for A549 cells, 16.79μg/ml for HepG-2 cells and 26.07μg/ml for SP2/0 cells, respectively and had no toxic effect to CEF. To evaluate antitumor activity in vivo of rGnRH-PEⅡ-luffinS fusion protein, animal models with solid tumor were established through inoculated SP2/0 subcutaneously. The tumor weight inhibition rate of rGnRH-PEⅡ-luffinS against SP2/0 was 50.3%, when administrated continuously for ten days with 100μg per mice. The result showed that the GnRH-PEⅡ-luffinS had antitumor activitiy both in vitro and in vivo.In conclusion, luffinP1 and luffinS2 were expressed in prokaryotic expression system and the protein biosynthesis inhibition activity of rluffinS2 was higher than rluffinP1 in rabbit reticulocyte lysate. The recombinant toxins GnRH-luffinS2 and luffinS2-GnRH were constructed, in which GnRH was as targeted moiety and luffinS2 was as toxic moiety. The cytotoxicity of these fusion proteins in vitro was low. Finally, PEⅡ, which has the function of translocation and proteolytic cleavage site, was inserted into GnRH-luffinS2, and GnRH-PEⅡ-luffinS was designed and expressed in prokaryotic expression system, the cytotoxicity in vitro and in vivo showed that the novel recombinant toxin GnRH-PEⅡ-luffinS had a potential to be used for targeted cancer therapy.更多还原