重组人源性抗HBsAg单链抗体在大肠杆菌中的表达、纯化与活性鉴定

【作者】 严兴；

【导师】 姚汝华；

【作者基本信息】 华南理工大学 ， 发酵工程， 2002， 硕士

【摘要】 乙型肝炎是一种世界范围的流行性传染病,但目前还没有有效的治疗手段,因此预防是防治乙型肝炎的重点。血源性抗乙肝表面抗原抗体可用于乙型肝炎的被动免疫,防止乙型肝炎的母婴垂直传播。但是,血源性抗体的来源有限且具有潜在的传染性,给它的应用带来限制。开发重组人源性抗HBsAg抗体,可以实现抗体的工程化大规模生产,而且使用安全,可以弥补血源性抗体的缺陷,取代血源性抗体,因此具有良好的社会效益和经济效益。 我们将针对HBsAg preS2区表位的人源性单链抗体基因转入大肠杆菌中表达,表达量可达菌体总蛋白的30%,并且目的蛋白在胞内形成包涵体。经Ni离子金属螯合亲和层析、凝胶层析两步纯化,重组抗体的纯度达到97%。将纯化后的单链抗体进行了复性。在优化复性方法后,在抗体浓度为0.34mg/mL时,蛋白收率可达50%。而且复性后的抗体具有较好HBsAg结合活性,其亲和常数达到0.23×10~8,同时测定了一株鼠源单克隆抗体的亲和常数,结果表明单链抗体的亲和力为一株鼠源单克隆抗体的十六分之一。竞争ELISA的结果表明,我们的单链抗体可以抑制一株鼠源单克隆抗体与HBsAg的结合,抑制率可达40.3%。并建立了测定单链抗体相对比效价的方法。 应用基质辅助激光解析飞行时间质谱(matrix assisted laser desorption ionization,MALDI-TOF-MS)精确测定了重组单链抗体的分子量,其结果只与理论值相差8道尔顿。同时还用DABITC/PITC双偶合法证实了重组单链抗体的N末端二个氨基酸序列与理论序列一致。另外,用等电点聚焦测定了单链抗体的等电点在7.3-8.1之间。 以上的研究结果表明,我们已构建成功的抗HBsAg单链抗体有望进行大规模生产,从而取代血源性抗体用于乙型肝炎的被动免疫。更多还原

【Abstract】 Hepatitis B is a world wide infectious disease, but up to now there has no effective methods to cure it, so prophylaxis of hepatitis B virus (HBV) is very important. The IgG anti-HBsAg derived from humam plasma can be used for the impassive immunization of HBV and prevention of HBV vertical transmission from mother to infant. But the IgG derived from human plasma is absent and has risk of transmisssion of infections. Therefore it is difficult to use it widely. Because human anti-HBsAg recombiant antibody can realize production of antibody in large scale and is very safe for clinical use, it will has good social effect and economic return that recombiant antibody takes place of the IgG derived from humam plasma. The single-chain antibody (scFv) gene that expresses anti-HBsAg preS2 epitope was transferered into E. coli. and was highly expressed in the host. The proportion of the protein of scFv to total protein of host can reach 30% and the recombinant scFv forms inclusion body in cells. The purity of 97% can be achieved after two step purification of scFv including Ni metal chelating chromatography (the first) and gel filtration (the second). And then the scFv in denaturant was renatured. By the optimized refolding method, the rate of recovery of protein is about 50% when the concentration of scFv is 0. 34mg/mL. The refolding scFv has strong ability to bind HBsAg, the affinity constant is 0.23 × 10~8, which is one of sixteenth of a murine Mab was detected at the same time. The result of competitive ELISA shows the scFv can repress the murine Mab and HBsAg to combine together, and the rate of repression can reach 40. 3%. A method to measure the relative potency of the scFv was established in the paper.We apply MALDI-TOF-MS to accurately measure the molecular weight of scFv, and there is only eight Dalton between the experimental result and theoretical volume. At the same time, we use DABITC/PITC method to confirm更多还原