【作者】 吕懿；

【导师】 郑金平；

【作者基本信息】 山西医科大学 ， 卫生毒理学， 2010， 硕士

【摘要】 目的：1.建立Arresten蛋白的JM109/pBV221/Arresten原核表达体系；2.研究Arresten蛋白的纯化方法,建立Arresten蛋白的制备工艺；3. Arresten蛋白纯品的药效学研究。方法：1.内源性血管生成抑制因子Arresten原核表达体系的建立从GenBank中获取Arresten基因序列,设计引物,在其羧基端加入六联组氨酸标签。从健康产妇胎盘组织中提取总RNA,经逆转录—聚合酶链式反应(RT-PCR)扩增出Arresten基因,将基因克隆入pMD19-T载体中,测序确认。经限制性内切酶EcoR I、Sal I双酶切,将Arresten基因定向插入表达载体pBV221,转入大肠杆菌JMl09进行温控诱导表达。经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫蛋白印迹技术I(Western blotting)对表达产物进行分析鉴定。2. Arresten蛋白的纯化超声破菌,分离得到包涵体,在变性条件下采用金属螯合亲和层析(MAC)方法纯化Arresten蛋白。使用Ni2+-NTA亲和柱,通过pH洗脱和咪唑洗杂结合的方法,对洗脱缓冲液和洗杂缓冲液进行优选,探寻Arresten蛋白的最优纯化条件,用BCA法测定纯化产物的蛋白含量,SDS-PAGE电泳检测纯化产物的纯度,并进一步对纯化产物复性。3. Arresten蛋白的药效学研究(1) Arresten蛋白作用于体外培养的人脐静脉内皮细胞(HUVEC)和人宫颈癌细胞(HeLa):MTT法检测Arresten蛋白对HUVEC和HeLa细胞增殖能力和黏附能力的影响；流式细胞仪分析Arresten蛋白作用下两种细胞凋亡的情况；细胞侵袭试验观察Arresten蛋白对两种细胞侵袭能力的影响。(2)鸡胚绒毛尿囊膜(CAM)实验观察Arresten蛋白对新生血管的抑制情况。(3)建立小鼠移植瘤模型,随机分组,治疗组瘤体内以3.2mg/kg剂量直接注射Arresten蛋白,对照组注射0.1ml生理盐水,每2天一次,共10天。观察瘤体大小变化、小鼠活动能力、饮食等状况。10天治疗后处死小鼠,摘取肿瘤,称量瘤重,计算肿瘤体积(tumor volume,TV)、相对肿瘤体积(relative tumor volume,RTV)及相对肿瘤抑制率。利用CD31抗体染色免疫组化方法检测肿瘤组织血管生成情况。结果：1.测序结果和酶切鉴定证实Arresten基因正确地插入表达载体中。经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,重组体Arresten在大肠杆菌JM109中获得表达,分子量约为26000,表达量约占菌体总蛋白的30%。经Western blotting检测表明该异源重组蛋白具有添加的亲和纯化标签His-tag的抗原活性。成功构建了有效的原核表达体系JM 109/pBV221/Arresten。2.通过对洗脱缓冲液和洗杂缓冲液的优化研究,发现采用含有15mM咪唑的洗杂缓冲液洗涤杂蛋白,能有效减少杂质蛋白的非特异性结合；调整洗脱缓冲液pH值为5.1,可使目的蛋白更好的洗脱下来。建立了最适宜于Arresten蛋白的纯化方法,可以一步得到纯度在97%以上的Arresten蛋白。3.浓度0.8μg/ml的Arresten蛋白能有效抑制HUVEC的增殖、侵袭(P<0.01),引起HUVEC的凋亡(P<0.01),并且在一定范围内随着Arresten蛋白浓度的增加,抑制作用与致凋亡作用明显加强。Arresten蛋白对HeLa细胞基本没有抑制增殖和致凋亡作用,有一定抑制HeLa细胞侵袭的效果(P<0.05),但没有明显的浓度依赖性。Arresten蛋白能明显减少HUVEC与HeLa细胞之间的黏附(P<0.01),0.8μg/ml的Arresten蛋白对其黏附抑制率为42.7%,再增加Arresten蛋白浓度,抑制率基本不变。4. Arresten蛋白能有效抑制鸡胚尿囊膜血管的生长(P<0.01),特别对中小血管的抑制作用较为明显。经Arresten蛋白作用,血管数目明显减少,血管辐辏低,以非特异性的杂乱、平行、贯穿等表现形式为主,出现血管细小稀疏、分支少、甚至血管断裂、毛细血管消失等现象。5. Arresten蛋白注射治疗组小鼠治疗前后饮食、行为无明显异常,反应能力、营养状况良好,Arresten蛋白在小鼠体内未引起明显免疫反应。在治疗第5天瘤体增长速度开始减慢,7天后肿瘤体积开始缩小。而阴性对照组小鼠随着时间延长,肿瘤呈进行性生长,饮食、活动和反应能力逐渐变差,后期呈恶病质态。治疗第10天Arresten蛋白组小鼠瘤重0.88±0.25g,瘤体积为765.19±249.41mm3,相对肿瘤体积为1.83±0.99,显著低于阴性对照组(P<0.01)。相对肿瘤抑制率为80.81%。经CD31多克隆抗体检测发现,治疗组瘤组织内血管形成明显减少。结论：本研究应用基因工程技术,在Arresten基因序列羧基端引入特异性纯化标签六联组氨酸标签,成功构建了原核表达体系JM109/pBV221/Arresten.通过对其纯化条件的研究,建立了纯度97%的Arresten蛋白的制备方法。生物学活性研究证明,纯品Arresten蛋白具有良好的抑制血管生成及抑制肿瘤浸润转移作用,对小鼠移植瘤有治疗效果。本研究是新型抗肿瘤制剂Arresten蛋白新药申报材料的核心组成部分,为Arresten蛋白的开发奠定了基础。更多还原

【Abstract】 ObjectiveTo explore the purification method of Arresten protein, in the same time to investigate the biological acticity of Arresten protein as the anti-tumor agent.Methods1. Expression of angiogenesis inhibitor Arresten in prokaryotic systemAccording to the mRNA sequence of Arresten gene from the GenBank, specific primers including the 6×His-tag (6 consecutive histidine residues) sequence in C-terminal for PCR were designed. Human Arresten gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from placenta tissue of a healthy puerpera, and cloned into the pMD19-T Simple Vector. The sequence of Arresten gene was analyzed. Digested with restriction endonuclease EcoR I and Sal I, inserted into plasmid pBV221. The constructed recombinant Arresten was transformed into E. coli JM109 and expressed primarily. The Western blotting method was used to examine whether the Arresten protein was expressed.2. Purification of angiogenesis inhibitor Arresten proteinPurification of Arresten protein was carried out with Ni2+-NTA agrose as there is a 6×His-tag at the C-terminal of the recombinant protein by immobilized metal affinity chromatography (IMAC). The His-Tag sequence binds to divalent cations (Ni2+) immobilized on NTA-based His-Bind resins. After unbound proteins are washed away, the target protein is recovered by elution with either imidazole or slight reduction in pH. Some factors influencing on the purification were discussed. This versatile system enables proteins to be purified under gentle conditions. The purification steps are wash the gel, binding of sample, wash the gel and elution sample. The purity and concentration were measured by 12% SDS-PAGE and Bradford method.3. The pharmacodynamics of Arresten protein(1) Arresten protein purified was incubated with HUVEC and HeLa cells in vitro:The inhibitory effect of Arresten protein on growth and adhesion of HUVEC and HeLa cells was examined by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis of HUVEC and HeLa cell was monitored by FCM. The migration of the cultured cells was examined by Boyden Chamber.(2) Chicken chorioallantoic membrane (CAM) angiogenesis model was used to identify the influence of Arresten protein on neovascularization.(3) To set up colorectal cancer model with C57BL/6 inbreeding line mice. Recombination Arresten protein was used to treat C57BL/6 mice bearing subcutaneously implanted primary SL4 fibrosarcomas.20 mice were divided into negative control group and Arresten protein group. Arresten protein at the dose of 3.2mg/kg was subcutaneously injected once per 2 days in the tumor of mice, whereas subcutaneous tumors were growing in the midline dorsum of each mouse. The mice of negative control group were injected physiological saline 0.1ml once per 2 days under the same conditions. To observe the activity, eating behavior and tumor growth of the mice. The two groups of mice were sacrificed after 10 day treated, then, tumor were taken off. The tumor volume and tumor weight were measured, then the inhibitory rate have been calculated. The microvessel densities of the tumors were measured by immunohistochemical staining with anti-CD31 monoclonal antibody.Results1. The sequence analysis and restriction endonuclease identification indicated that the Arresten gene was inserted into the expression vector ssuccessfully. SDS-PAGE analysis indicated that expressed product was about 26000 and its amount was about 30% of total bacterial proteins. Western blotting indicated that the protein could react with His-tag antibody.2. The unbound proteins were washed away effectively with wash buffer including 15mM imidazole, and the target protein is recovered by elution in elution buffer pH value as 5.1. Under the condition, the expressed protein was one-step purified to 97% using Ni2+-NTA affinity chromatography method, and with a yield of 172.4μg/mL. The renaturation of Arresten protein was finished successfully by means of gradient dialysis.3. Arresten protein (0.8μg/ml) inhibits the proliferation and migration of HUVEC and promotes its apoptosis effectively; and the inhibition was dose-dependent. However, it had no significant effects on proliferation and apoptosis of HeLa cells. The migration of HeLa cells was also inhibited by Arresten protein, but increasing the protein dose, the inhibition was at a same level. Arresten protein could markedly decrease the adhesion rates of HUVEC and HeLa cells, and their adhesion inhibition rate reached to 42.7%. 4. Compared with PBS group, the vessels of Chicken chorioallantoic membrane (CAM) in Arresten protein (3.2μg/ml) treated groups were obviously damaged. So Arresten protein call significantly inhibit angiogenesis of CAM.5. The Arresten-treated group mice showed no weight loss or unusual behavior during the course of treatment and the growth of tumor was significantly slow; but the negative control group mice showed the expression that was angular reach cachexia to wait and tumors grew rapidly. Arresten protein evoked obvious antitumor effects when it was given 5 days. After treatment, compared with the control group (3461.20±927.53mm3, n=9), the average tumor volume of the Arresten-treated group mice was reduced in size (765.19±249.41mm3, n=10, P<0.01) with a relative inhibition rate of 80.81%. While the tumor weight also decreased (0.88±0.25g, n=10, P<0.01) in comparison with the control group (2.16±0.37g, n=9). To evaluate the suppressive effect of Arresten on tumor angiogenesis, tumor tissue sections were immunohistochemically stained with an endothelial specific antibody factor and microvessels in tumor tissues were randomly counted. A dramatically decreased microvessel density in tumor tissues was revealed in the Arresten-treated mice compared with control tumor tissues treated with physiological saline.ConclusionThrough of the gene engineering, the 6×His-tag (6 consecutive histidine residues) sequence was introduced in C-terminal of Arresten gene. The prokaryotic expression recombinant pBV221-Arresten was construct successfully and fusion protein was expressed in E. coli JM109. The optimal conditions were investigated, and the best conditions for separation and purification were established. Study on biological acticity shows that the Arresten protein can inhibit efficiently angiogensis and the growth and metastasis of tumor. Arresten protein is a powerful angiogenic inhibitor and is believed to have therapeutic potential in the treatment of solid tumors. The research may provide a theoretical basis for developing a new antitumor drug.更多还原