【作者】 杨奎；

【导师】 卢圣栋；

【作者基本信息】 中国协和医科大学 ， 生物化学及分子生物学， 2003， 博士

【摘要】 已知共有上千种抗肿瘤或抗病毒活性的人体异源蛋白，但在付之临床应用时存在两个问题：一是无特性（或靶向性）地对所有细胞的杀伤作用导致的严重毒副作用问题；二是作为人体异源蛋白质，在导入人体血循环时出现的严重的免疫原性问题。为解决上述问题，上个世纪80年代曾出现过利用肿瘤细胞受体配体基因与毒素基因Ⅱ、Ⅲ功能域编码基因构建的融合基因及其所产生的导向融合蛋白。但临床研究表明，这种导向蛋白虽能在一定程度上增强了毒素蛋白质的导向性与特异性，但不完全。与此同时，毒素蛋白质在这种情况下，其强烈的免疫原性问题依然存在。在临床应用时，虽初期有良好疗效，但在4周之后，因抗体形成，发生中和效应而完全失效。虽然通过多种尝试来修饰或封闭毒素的抗原决定簇，如PEG修饰和点突变改造毒素的氨基酸组成，但均因修饰而导致毒素失活，或不能完全消除毒素蛋白的免疫原性，最终还是导致了毒素的失效。因此，寻找一种安全、有效地导入靶细胞，从而杀灭肿瘤靶细胞而又避免毒素蛋白对正常细胞的毒副作用与免疫原性的方法，是必要的。多数腺癌细胞的表面会特异性地表达促性腺激素释放肽（GnRH）受体，使用可以与其特异性结合的GnRH作为靶向蛋白，将有杀伤作用的绿脓杆菌毒素导入肿瘤细胞，可望杀伤肿瘤细胞。这一设计基本解决了毒素导向杀伤肿瘤细胞问题，但因一些正常细胞表面也表达GnRH受体以致也还可能把毒素导入部分正常细胞而出现毒副作用问题，与此同时，绿脓杆菌毒素对于人体来说是一种异源蛋白，将会产生免疫原性，为解决准确导向完全避免毒素的毒副作用问题与完全避免异源毒素的免疫原性问题，本课题设计了一套非病毒载体靶向杀伤肿瘤细胞的基因治疗方案。把原来以毒素蛋白质导入血循环的形式改为以编码毒素蛋白的基因重组体（DNA）导入血循环的形式，即利用非病毒载体将对细胞有强烈杀伤作用的PEⅢmut（绿脓毒素第三区段的突变基因片段）以表达型重组体的形式靶向导入肿瘤细胞，致使PEⅢmut仅在肿瘤细胞中表达，从而杀灭该细胞。同时，利用肿瘤细胞是缺氧细胞的特点，以缺氧条件作为基因可控表达的控制条件。这样，既避免了毒素蛋白的免疫原性，也避免了对任何其他类型细胞的杀伤作用。非病毒载体部分的设计：首先，利用PCR技术得到靶向蛋白GnRH的基因和DNA结合蛋白SON的72个氨基酸基因，重组构建成融合蛋白GnRHSON的融合编码基因。将该序列插入酵母表达载体pPIC9K中，质粒线性化后电转甲醇营养型酵母KM71，利用G418筛选和小规模诱导培养筛选高表达株。经激光密度扫描，表达量为1 mg／L，在培养上清总蛋白中占58.5％。对此高表达株进行大规模培养诱导，用硫酸铵沉淀法分离纯化蛋白，并通过透析、浓缩回收到纯度较高的蛋白。因为酵母表达载体表达的蛋白量很低，所以进一步构建大肠表达载体的克隆。在克隆过程中，首先考虑是将所有编码的氨基酸换成大肠嗜好的密码子；并且考虑到毒素基因要在细胞核内表达，在导向蛋白后接入核定位序列（NLS），使DNA结合蛋白携带编码毒素的DNA穿过核孔，进入核内进行转录，其后在核糖体翻译成蛋白质。把优化后的GnRH-TAP-SON分别插入一系列表达载体，在P1噬菌体Ref基因后进行融合表达。考虑到凝血酶切割有问题，在Thrombin后加丝氨酸编码，但切割的过程中发现，切割后的两个蛋白分子量比较接近，并且都富含阳性氨基酸。因此改变设计，提出在GnRH后接入易于表达的TNFmut基因的一个片段（无活性且无结合TNF受体能力），同时考虑到TAP可能影响了表达，还克隆了GnRH-TNF-SON。结果这两个蛋白都得以表达。我们选择GnRH-TNF-TAP-SON进一步大瓶表达和纯化。提取包含体，经7M盐酸胍溶解，经过反相柱、离子柱和疏水柱纯化后，电泳银染为一条带，以此作为非病毒载体。作为基因治疗的功能基因PEⅢmut的构建和纯化：以转染真核细胞的载体pGL3为骨架，用经过改造的绿脓杆菌毒素第Ⅲ片段PEⅢmut取代荧光素酶基因，得到pGL3-SV40-PEⅢmut。同时利用实体瘤处于缺氧状态这一特性，本课题设计了缺氧反应元件（hypoxia-responsive element，HRE）作为毒素基因表达的增强子，将4拷贝的HRE插入SV40启动子的上游，构建了pGL3-4×HRE-SV40-PEⅢmut。为了使PEⅢmut只在缺氧条件下（肿瘤细胞内）表达，我们构建了pGL3-4×HRE-miniCMV-PEⅢmut。这里，HRE只能在缺氧条件下才能发挥对miniCMV的驱动作用，而miniCMV只有在HRE的驱动下才能发挥启动作用，从而实现了准确杀伤的靶向设计。又在启动子后插入一段兔内含子intronⅡ以提高其下游基因的表达，成为pGL3-4×HRE-miniCMV-intronⅡ-PEⅢmut，以便提高PEⅢmut的表达效率。质粒DNA的纯化过程包括：初步收集质粒DNA；经CaCl₂沉淀去除部分RNA和基因组DNA；第一根DEAE阴离子柱捕获核酸；再经第二根阴离子柱resourceQ柱进一步纯化。最终超螺旋DNA的量经电泳检测达90％以上，蛋白电泳和BCA法均未检测到蛋白。将DNA与蛋白溶液按不同比例混合（1：1，2：1，4：1，8：1），在0.8％琼脂糖电泳凝胶上观察电泳迁移率变化，其中1：1混合后的滞留结果最好。DNA与蛋白复合物在4℃放置一个月，0.8％琼脂糖电泳检测，DNA仍几乎完全滞留在加样孔中，说明形成复合物后在4℃可以稳定保存。分别用脂质体和我们纯化后的融合蛋白非病毒载体分别转染编码绿色荧光蛋白和荧光素酶的报告基因重组体质粒，在荧光显微镜下观察均有绿色荧光蛋白表达，荧光发光仪检测荧光素酶表达，二者的值比较接近，说明我们设计的融合蛋白非病毒载体及蛋白质-DNA复合物转染肿瘤细胞的实验设计路线是可行的。在有氧条件下，融合蛋白非病毒载体与pGL3-SV40-PEⅢ复合物对GnRH阳性的细胞在8ug／ml剂量组与对照组OD值相比，P＜0.05，均有显著性差异，说明均有显著的杀伤作用。对GnRH阴性对照细胞无杀伤作用，P＞0.05。对GnRH阳性的人胚肾HEK293、肝癌HepG2、宫颈癌Hela和乳腺癌MCF-7的杀伤率（1-存活率）分别为72.9％、57.9％、54.1％和51.7％。由此证明了GnRH具有很好的导向性。但这一结果同时说明，这种在有氧条件下出现的强烈的杀伤作用亦可发生在其表面有GnRH受体的一切非肿瘤的正常细胞方面。因此，这个设计方案对临床应用研究是不完善的，可能出现毒副作用。而这一结果，则可以作为下述设计的一个参考。非病毒载体蛋白与pGL3-4HRE-miniCMV-PEⅢ复合物在有氧条件下，对GnRH阳性细胞即使在8ug／ml剂量组与对照组OD值相比，P＞0.05，没有显著性差异，说明无杀伤作用。在缺氧条件下，非病毒载体蛋白与pGL3-SV40-PEⅢ复合物对GnRH阳性的细胞在8ug／ml剂量组与对照组的OD值相比，P＜0.05，均有显著性差异，说明有杀伤作用。对GnRH阳性细胞肝癌HepG2、宫颈癌Hela和乳腺癌MCF-7的杀伤率（1-存活率）分别为61.3％、60.1％和53.9％。这一结果进一步说明，利用SV40启动子的设计是不理想的、甚至是不可取的。只有实现功能基因可控表达设计，才可实现准确的靶向杀伤设计。非病毒载体蛋白与pGL3-4HRE-miniCMV-PEⅢ对GnRH阳性细胞在8ug／ml剂量组与对照组OD值相比，P＜0.05，具有显著性差异。对HepG2、Hela和MCF-7的杀伤率（1-存活率）分别为57.1％、56.9％和41.7％。流式细胞仪检测pGL3-HRE4-miniCMV-PEⅢ蛋白复合物在低氧条件下发生了细胞的凋亡。细胞活性试验的结果表明，本研究所设计的靶向蛋白可以有效地介导毒素基因进入富含特异性受体GnRH的肿瘤细胞，并表达毒素蛋白，从而杀伤肿瘤细胞，但将会有比较严重的毒副作用，而只有在缺氧状态下，低氧应答元件与miniCMV作为“第二开关”具有很好的选择性，使毒素基因仅在低氧的环境下表达，发挥准确特异靶向性杀伤细胞表面有GnRH受体的恶性肿瘤细胞。既不杀伤细胞表面无GnRH受体的正常细胞，也不杀伤细胞表面有GnRH受体的非缺氧正常细胞。这是因为即使上述毒素基因被导入正常细胞，因其为有氧细胞，毒素不表达。这样的“双开关”设计，即利用受体配体的关系的导向设计，同时利用缺氧条件作为基因可控表达设计，则实现了对一类肿瘤准确的靶向基因治疗设计。把毒素基因用于这种可控表达设计是我们的首创。这个设计要求“个体化诊断与个体化治疗”。因为该复合药物只适合于GnRH受体阳性的肿瘤细胞，如GnRH受体阳性的肝癌、乳腺癌、结直肠癌、胰腺癌、卵巢癌、前列腺癌、垂体瘤等。本课题仅仅初步探索了以“个体化诊断与个体化治疗”为特征的21世纪新医学，在一个点上展开的情况，参与了相关的正反两个方面经验的积累。更多还原

【Abstract】 It is known that thousands of naturally occurring toxin proteins possess anticancer activity. But few of them were applied in clinical research, and none was successful. The obstacles exist in two aspects, nonspecificity and immunogenicity. The toxins administrated in vein leads to the production of antibody against itself, thus the neutraliztion response later limits the cure effect of toxin, even leads to the invalidation. The targeting fusion protein with toxin was constructed appeared in last century consist of ligand to receptors on tumor cells and function domains of toxins. They are constructed with the technique of DNA recombination, and expressed fused. Targeting toxin could limit the toxicity in cells with corresponding receptors, but the toxicity to normal cells with the same character could not be avoided. The immunogenicity could not be eliminated either.Aiming to the obstacles against application of toxins in tumor therapy, we proposed a design, to avoid not only the immunogenicity but also the toxic side effect. In this project the expression recombinants with coding sequence of PEIIImut toxin protein. A kind of fusion gene was done and expressed. Its fusion protein contained GnRH as the targeting molecule and partial sequence of Son as DNA binding polypeptide. Toxin expression recombinant could be packed by the SON domain of fusion protein, and was led to the one kind of cancer cells with the guidance of GnRH. In cells with GnRH receptors, toxin protein were expressed, and cancer cells were killed by cytotoxic function. On the basis of the hypoxia specialty of tumor cells, hypoxia response element was applied to regulate the expression of toxin gene. Therefore the normal cells with GnRH receptors could not be killed since they are aerobic. Thus, both the immunogenicity and the toxic side effect would be avoided in this design.The coding sequence of GnRH was obtained by PCR, and so was the 72 amino acids of Son. The fusion sequenc of GnRHSON was inserted in pPIC9K of pichia pastoris expression system. Linearized recombinant was transformed into KM71 with use of electroporation. Using G418 growth inhibition for screening, some strains showed high expression of GnRHSON. The yields were about 1mg/L, and the target protein secreted into the culture medium reached 58.5% of total supernatant proteins. The strain giving the highest expression level was inoculated and induced to harvest the target fusion protein. Then it was purified by precipitation of ammonium sulfate, dialysis, concentration in succeeding procedures. To enhance the expression level, the expression system was switched to E. coli, and the codens of fusion protein were changed to the ones E. coli preferring. To improve the transfering and expression efficiecy in vivo, one kind of nuclear locating sequence, TAP, was inserted between GnRH and SON. Then the coding sequence of GnRH-TAP-SON was cloned into several vectors, and expression was obtained by recombinant pCW-GnRH-TAP-SON, in which target protein was fused to Ref, a P1 phage gene. Thrombin recognizing sites were added to the N terminal of target sequence to make the cleavage possible. But the fragments caused after cleavage were too resembling to separate. They got the same electrical property, and the molecular mass were closed nearly. So another recombinant was constructed, pCW- GnRH-TNF87-TAP-SON, and the protein was expressed as inclusion body. The purification procedure included reverse phase column, cation exchange column and hydrophobic interaction column. The purity of the target protein was tested by silver staining after SDS- PAGE electrophoresis, and only one bind appeared.The toxin gene used in this project was the mutant coding sequence of the third domain in PE. The last four amino acids were replaced as KDEL to enhance the toxicity. Four-copy HRE was inserted into the upstream of CMV promoter, and the promoter was also deleted to be its mini form. To enhance the activity of the promoter, intronⅡin globin of rabbit was inserted between miniCMV and toxin gene. The toxin expression recombinant was purified by chromatography, after precipitated by CaCl₂, first using the DEAE anion ion exchange column, second using Resource Q column. The supercoiled form accounted to be 90％at last. Protein couldn’t be detected by SDS-PAGE electrophoresis and BCA kit.The solution of target protein and DNA was mixed at different ratios （1: 1, 2: 1, 4: 1, 8: 1）. Electrophoresis of agarose gel showed 1: 1 was the best. DNA-protein complex was stored at 4℃one month, electrophoresis result showed DNA was almost detained in sample hole. It illustrated complex was stored steadily at 4℃.The recombinant plasmids coding green fluorescence protein（GFP） and luciferase reporter gene were tranfected by lipofectin and purified fusion protein as nonviral vector, respectively. Green fluorescence protein and luciferase were both expressed. The results showed that fusion protein as nonviral vector and protein-DNA complex transfected tumor cells was effective.Cell lines expressed GnRH receptor HEK293（human embryonic kidney, normal cell line）, HepG2 （Hepatocarcinoma）, Hela（cervix carcinoma） and MCF-7（mammary cancer）, and cell line 2BS （human embryonic lung） without GnRH receptor expression were used in the targeting killing experiment. Under aerobic condition, pGL3-SV40-PEⅢ/protein GnRH-TNF87-TAP- SON complex could kill all of the receptor positive cells strongly, but could not kill the negative cells such as 2BS. When dose of complex was 8 ug/ml, the killing rates were 72.9％, 57.9％, 54.1％and 51.7％corresponding to HEK293, HepG2, Hela and MCF-7. The results suggested that the fusion protein GnRH-TNF87-TAP-SON could lead the toxin recombinant into all GnRH receptor positive cells including of normal GnRH receptor positive cells. Thus, the project maybe lead side effect and was not consummate. Under the same condition, nonviral vector and pGL3-4HRE-miniCMV-PEⅢcomplex showed no killing effect on HEK293. So the toxic side effect could be avoided by the control of GnRH and HRE- miniCMV.Under the hypoxia condition, using nonviral vector protein GnRH-TNF87-TAP-SON and pGL3-SV40-PEⅢat the dose of 8 ug/ml could kill the positive GnRH receptor cells such as HepG2, Hela and MCF-7, and the killing rate was 61.3％、60.1％and 53.9％, respectively. It further showed SV40 promoter was not desirable. Function gene controllable expression was designed, then accurate targeting killing could be come true. Nonviral vector protein GnRH- TNF87-TAP-SON and pGL3-HRE4-miniCMV-PEⅢat the dose of 8 ug/ml could kill the positive GnRH receptor cells such as HepG2, Hela and MCF-7, and the killing rate was 57.1％, 56.9％, 41.7％, respectively.Under the hypoxia condition, nonviral vector protein GnRH-TNF87-TAP-SON and pGL3-HRE4-miniCMV-PEⅢcould lead cell apoptosis through flow cytometer analysis.Fusion protein designed in this project could mediate toxin gene into the GnRH-R positive cells, the toxin protein expressed and killed the cells. With the control by HRE- miniCMV, the expression of toxin was limited only in hypoxia GnRH-R positive tumor cells, the normal cells with GnRH-R could not express the toxin protein due to its aerobic characteristics, and could not be killed. Thus, normal cells without GnRH-R and the normal cells with GnRH-R could not be killed. Because the toxin gene could not be expressed in normal aerobic cells. The bi-switch design utilized both relationship of receptor and ligand and hypoxia condition as controllable gene expression, then the gene therapy design of accurate targeting killing tumor cells could be come true. Toxin gene used in the controllable expression design was first.The design demands individual diagnosis and individual treatment. Because the complex drugs only fitted to hypoxia tumor cells with positive GnRH receptor, such as Hepatocarcinoma, mammary cancer, colorectal tumor, pancreatic cancer, ovary tumor, prostate carcinoma and pituitary tumor. It is the preliminary exploration of Medicine in 21 century, characterized as "individual diagnosis and individual treatment".更多还原