【作者】 姜静；

【导师】 王长海；

【作者基本信息】 大连理工大学 ， 生物化工， 2011， 博士

【摘要】 通过肿瘤特异靶向肽将药物输送至肿瘤靶向部位进行肿瘤治疗已成为一个重要的癌症治疗策略,尤其是选择性作用于特定靶点的高效、低毒、特异性强的抗肿瘤分子靶向药物的研发已成为抗肿瘤靶向研究领域中的重要研究方向。白细胞介素-2(IL-2)是一种多功能细胞因子,具有刺激细胞毒T淋巴细胞分化、激活细胞毒T淋巴细胞、自然杀伤细胞、嗜酸性细胞、肥大细胞的非特异性杀伤作用以及增强B淋巴细胞的抗体分泌和增殖等生物功能。目前临床上主要将其用于免疫佐剂调动机体的系统免疫,达到辅助治疗控制肿瘤的目的；但临床试验表明IL-2单药治疗癌症的有效率低,副作用大。为克服这个问题,本文选择了具有肿瘤靶向作用的生长抑素类似物奥曲肽,运用基因工程手段与白细胞介素-2融合,构建了一种全新的生长抑素受体靶向融合蛋白(somatostatin receptor target interleukin 2, SIL)。将奥曲肽-白细胞介素-2融合蛋白(SIL)基因进行克隆,插入pET-25b载体,并转化到大肠杆菌BL21(DE3),获得表达菌株BL21(SIL2),经PCR扩增筛选到阳性重组表达菌株后在摇瓶中进行试表达；经IPTG诱导后,筛选到目的蛋白表达率高的工程菌株。在20L发酵罐中对该菌株的培养基及发酵工艺进行了优化,在温度37℃,pH 7.2,IPTG的浓度为10 mg/L(在菌体密度OD600达到20左右时加入)、诱导时间为4～5h的发酵条件下,细胞的培养密度(OD600)和细胞内目的蛋白表达率分别达到60和30%以上。采用酶法(0.5～1‰溶菌酶、4‰脱氧胆酸)与高压匀浆相结合的方法进行破菌；并对细胞内包含体的洗涤工艺及洗涤缓冲液组成进行了优化,进而获得了较高纯度的SIL包含体蛋白。采用亲和层析对包含体进行纯化并经稀释复性获得具有天然结构的SIL蛋白。样品经8 M urea尿素进行变性后,在填料为Ni-NTA Super Flow、上样量为每ml填料10mgSL、pH值梯度洗脱(pH 4.5一步洗脱)的层析条件下,SIL变性蛋白纯度达到93.0%以上；纯化后的SIL经稀释后在5 mM DTT、2 mM GSH的氧化-还原体系中直接进行氧化还原,在2μM CuSO4的催化作用下稀释尿素浓度至4M的条件下,达到复性目的,复性蛋白的纯度和复性收率分别达到95%和20%以上；经刺激CTLL-2细胞增殖法测定,复性后的SIL蛋白与IL-2的比活接近。经过对不同肿瘤细胞生长抑素受体的检测、体外Co-IP实验、免疫荧光组化、体外细胞结合实验以及体内分布实验,证明所设计的靶向融合蛋白具有与特定肿瘤细胞的靶向结合作用；与IL-2相比,靶向SIL能够结合到具有生长抑素受体Ⅱ(SSTR2)的肿瘤细胞上。体外细胞结合实验结果表明SIL结合到肿瘤细胞后仍能够发挥IL-2刺激CTLL-2细胞的增殖作用；体内125I-SIL药代分布实验结果表明,SIL在体内具有肿瘤靶向分布的优势,从2-4 h之间肿瘤与其他组织的放射性相比明显增高,T/NT大于1。通过建立微血管渗漏模型,以Evan’s blue渗漏量作为检测指标,在荷瘤小鼠C57上实施SIL及IL-2诱发的微血管渗漏综合症(Vascular Leak Syndrome, VLS)实验；结果表明,SIL诱发明显VLS的剂量与IL-2无明显差异。在H22荷瘤的昆明小鼠模型以及H22荷瘤C57小鼠的荷瘤模型中进行了SIL和IL-2的体内药效学研究,确定了SIL与IL-2的药效差异剂量,在2.5×104IU/kg治疗剂量下,SIL抑瘤率与空白对照组和IL-2低剂量组相比差异显著(P<0.01 vs. control; P<0.05 vs. IL-2 low);在C57荷瘤小鼠的生命延长率实验中验证了同样的结果,低剂量SIL组的小鼠存活率最高,停药10d后存活率100%,IL-2组存活率仅为50%。更多还原

【Abstract】 Cancer immunotherapy by means of anti-tumor monoclonal antibodies, cell adoptive immunotherapy, cancer vaccine has become one of the most important strategies of tumor target therapy. Interleukin 2 (IL-2) is a cytokine with a variety of biological functions, also can enhance the B lymphoid antibody secretion and cell proliferation, which is mainly used as an immune adjuvant to mobilize the body’s immune system to control tumor in the clinic. Although the systemic administration of IL-2 was shown to stimulate antitumor responses in vivo, its efficacy has been limited by the development of serious side effects and low efficacy in the clinic. To overcome the problem, we focus on tumor targeting somatostatin analogue, octreotide, which has been researched for tumor treatment and diagnosis purpose, and engineer a novel fusion protein with the chimeric functions of somatostatin receptor targeting interleukin 2 (SIL).In this research, the fusion protein gene composed octreotide and interleukin 2 DNA sequence was successfully constructed into pET-25b vector and transformed into E.coli BL21 (DE3) to obtained the expression strain. Based on the studies of laboratoty scale, the optimized of culture medium and the scale up conditions of fermentation were developed in the bioreactors of 20L. Under the conditions of 37℃, pH 7.2, and induced with isopropy-β-D-thiogalactoside (IPTG) at the concentration of 10 mg/L when OD600 gets 20, the protein expression level beyond 30% with the final OD600 at 60.During inclusion body washing process, the cells were broken by method of enzyme lysis (0.5 %o～-l%o lysozyme,4%o deoxycholate acide sodium) combined with high pressure homogenization. In the system of 8 M urea,5 mM DTT and pH 8.6, the proteins were denatured, and the purity above 93% of SIL protein was obtained by Ni-NTA Super Flow purification and the binding capacity of Ni-NTA media for SIL protein was optimized to 10 mg/ml. The denature protein was diluted and oxidized in refolding buffer containing 4M urea, 2mM GSH-2 u M CuSO4, and the purity and yield of refolded protein were arrived to 95% and 20% respectively during refolding process.As for the tumor targeting confirmation, a serial of experiments were carried out including somatostatin receptors detection of various tumor cells, Co-immunoprecipitation experiment, immunofluorescence staining and cell binding assays in vitro. From in vitro test, it showed that SIL was still able to stimulate the proliferation of CTLL-2 cells after binding on the the tumor cells via somatostatin receptor 2 (SSTR2) that to be determined expressed on the tumor cells. In addition, the in vivo distribution of 125I-SIL in tumor burden nude mice for pharmacokinetic study showed that distribution of 125I-SIL in tumor tissue was significantly higher than other tissues, and the T/NT was greater than 1.Using the microvascular leakage (VLS) model, Evan’s blue leakage was adapted as specification to determine the degree of VLS induced by IL-2 or SIL. The experiments were carried on C57 tumor burden mice by injection of SIL and IL-2, and the results manifested that there was no significant deviation between SIL and IL-2 on inducing significant VLS. Anti-tumor study in vivo has determined the dose that IL-2 and SIL exhibit discrepancy on anti-tumor effects. Under the treating dose of 2.5×104 IU/kg, SIL exhibits significant anti-tumor effect compared with the control group and the IL-2 group (P<0.01 vs. control; P <0.05 vs. IL-2 low). Life-extension experiments on H22 tumor burden C57 mice demonstrate that 10 days survival rate after stopping treating was 100% for low dose treating group of SIL, while IL-2 treating group survival rate was only 50%.更多还原