人脂联素球状区大肠杆菌表达、纯化及单克隆抗体制备

发布时间：2019-03-15 14:08

【摘要】：随着科学技术的飞速发展，人们的生活水平也有了很大提高。特别是二战后，人们的饮食结构有了很大变化，肉类和油脂的消费剧增是食物结构中最大的变化。而与之相随的是，糖尿病、心脑血管疾病、肿瘤等的发病率也飞速增多。中国已经是肥胖人口增长最快的国家之一，是糖尿病患者人数居世界第二，Ⅱ型糖尿病发病率世界第一。研究表明，血浆中脂联素水平与这些疾病的发生有密切关系，且脂联素球状区是其行使功能的主要结构。故通过对脂联素水平的测定可以为临床诊断这些疾病及临床用药提供指导。本研究利用基因工程技术获得具有生物活性的人脂联素球状区融合蛋白，利用其制备单克隆抗体，为建立人脂联素免疫学检测方法奠定基础。 根据NCBI上公布的人脂联素基因序列NM_004797.2，分析其密码子并对其密码子进行优化，然后人工合成人脂联素基因球状区序列，通过酶切，构建表达载体pET41a-gAd。在大肠杆菌中表达，通过调节诱导剂浓度、诱导温度和诱导时间进行优化，确立最佳诱导条件为：37℃，IPTG浓度0.1mmol/L，诱导表达6h。表达出人脂联素球状区融合蛋白，用Ni亲和层析柱纯化，采用包涵体复性技术对表达蛋白进行复性，获得具有生物活性的重组蛋白。把纯化的人脂联素球状区蛋白以适当剂量免疫BABL/C小鼠，取免疫效价较高小鼠的脾细胞和骨髓瘤细胞进行细胞融合。经HAT培养筛选、有限稀释法克隆化，获得能分泌抗人脂联素球状区单克隆抗体的细胞株。对细胞株进行扩大培养，收集杂交瘤细胞，，接种BABL/C小鼠腹腔诱生腹水，并对腹水进行纯化，经SDS-PAGE分析其纯度，并鉴定其亚型。 本研究成功构建了pET41a-gAd载体，能高效表达出具有生物活性的人脂联素球状区融合蛋白；该蛋白分子量约为45kD，经纯化后纯度为90%。用此蛋白成功制备备单克隆抗体。获得了人脂联素球状区单克隆抗体细胞株1株，腹水纯化后单抗纯度为91%，所获的单克隆抗体亚型为IgG1。
[Abstract]:With the rapid development of science and technology, people's living standards have been greatly improved. Especially after World War II, people's dietary structure has changed greatly. Consumption of meat and fat is the biggest change in food structure. With it, the incidence of diabetes, cardiovascular and cerebrovascular diseases, tumors and so on also increased rapidly. China has one of the fastest-growing obese people, the world's second largest number of people with diabetes, and the world's highest incidence of type 2 diabetes. The results showed that the level of adiponectin in plasma was closely related to the occurrence of these diseases, and adiponectin globular region was the main structure of its function. Therefore, the determination of adiponectin level can provide guidance for clinical diagnosis of these diseases and clinical use of drugs. In this study, human adiponectin globular fusion protein with biological activity was obtained by genetic engineering technique, and monoclonal antibody was prepared, which laid a foundation for the establishment of immunologic detection method of human adiponectin. The codon of human adiponectin gene was analyzed and optimized according to the published human adiponectin gene sequence NM_004797.2, in NCBI. Then the spherical region sequence of human adiponectin gene was synthesized and the expression vector pET41a-gAd. was constructed by restriction enzyme digestion. The expression in E. coli was optimized by adjusting the concentration of inducer, inducing temperature and inducing time. The optimal induction conditions were 37 鈩

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