丁型肝炎病毒抗原的原核表达纯化与血清学检测方法的建立

发布时间：2018-01-20 01:38

  本文关键词： 丁肝抗原 HDV 血清学检测 原核表达　出处：《中国疾病预防控制中心》2013年硕士论文　论文类型：学位论文

【摘要】：[目的]Hepatitis D virus (HDV)可以引起丁型病毒性肝炎。HDV病毒的核蛋白即为丁肝抗原,可以刺激机体产生特异性的抗体。现在很多实验室使用重组生产的丁肝抗原作为标准抗原,对丁肝患者进行血清学检测。获取高纯度的丁肝抗原是本实验的第一个目的。并通过对表达纯化资料的积累,为下一步的中试生产做准备。当前还没有我国丁肝流行病学的准确资料,主要是因为没有统一的检测标准和检测试剂。使用本实验生产的丁肝抗原建立起特异性和敏感性均较好的血清学检测方法是本实验的第二个目的。将其应用于疾控工作,得到我国丁肝流行病学的准确资料。 [材料和方法]本实验在Genbank数据库获取了HDV的序列信息,选择HDV的S-HDag的编码序列,在大肠杆菌偏好密码子的基础上,进行了密码子的替换。预测了该抗原mRNA的二级结构,对影响表达的序列通过简并密码子进一步优化后,人工合成该基因。通过基因工程技术,双酶切后,连接至pET43.1.a表达载体,在大肠杆菌BL21(DE3)工程菌株中进行表达。通过控制表达条件,最终确定的表达程序为：标准转化程序获取的单克隆接种5m1小试管,37摄氏度过夜培养后,1：50稀释接种于250m1锥形瓶中,37摄氏度生长至OD值约为0.8,使用诱导剂为ITPG,终浓度为1mM/L,37摄氏度下诱导3h,最终获得HDV抗原的可溶性表达。使用的培养基为LB培养基,选择性的抗性为氨苄青霉素,使用终浓度为50mM/L。 获得的大肠杆菌菌体,使用超声破碎仪,完全破碎菌体,在10000rpm下离心,保留上清,在1M/L的NaCl条件下,利用表达目的蛋白的His-tag,在镍离子和咪唑的作用下,通过亲和层析初步获得了目的蛋白,后进一步经过离子交换层析和分子筛层析,最终获得了高纯度的蛋白,符合血清学检测试剂盒应用的标准。 将本实验获取的目的蛋白包被ELISA板,包被的条件是在pH9.5的包被碳酸缓冲液作用下,每孔2-4ng,4摄氏度过夜,组装成检测试剂盒,通过间接法测定标本中的IgM抗体,根据结果对其进行判定。对来自丁肝血清盘的标本和临床收集的血清标本进行检测,该方法的特异性和敏感性均较好。 [结论]本实验获得了具有抗原活性的目的蛋白,建立了血清学检测方法。通过基因工程技术,表达目的蛋白,为下一步的中试生产做好了技术准备。低成本的优势和良好的效果可以为该检测方法的市场化做铺垫,进而确定统一的标准和试剂,从而得到我国准确的流行病学资料。
[Abstract]:[Objective] the nucleoprotein of Hepatitis D virus can cause hepatitis D. the nucleoprotein of HDV is liver D antigen. It stimulates the body to produce specific antibodies. Many laboratories now use recombinant hepatitis D antigens as standard antigens. It is the first aim of this experiment to obtain high purity of hepatitis D antigen by serological examination in patients with hepatitis D. and through the accumulation of purified data of expression. To prepare for the next pilot-scale production. At present, there are no accurate data on the epidemiology of hepatitis D in China. The main reason is that there is no uniform detection standard and reagent. It is the second purpose of this experiment to establish a specific and sensitive serological detection method using the antigens produced in this experiment. Control work. The accurate data of hepatitis D epidemiology in China were obtained. [Materials and methods] the sequence information of HDV was obtained from Genbank database and the coding sequence of S-HDag of HDV was selected based on the preference codon of Escherichia coli. The secondary structure of the antigen mRNA was predicted. After further optimization of the sequence affecting the expression by degenerate codon, the gene was synthesized artificially. Ligated to pET43.1.a expression vector and expressed in Escherichia coli BL21DE3 engineering strain by controlling the expression conditions. The final expression procedure was as follows: 1: 50 was diluted into 250m1 conical bottle after overnight culture with monoclonal inoculation of 5m1 small test tube and 37 degrees Celsius by standard transformation procedure. 37 degrees Celsius grew to an OD value of about 0.8, the inducer was ITPG.The final concentration was 1 mm / L ~ (37 鈩，

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