Engineered polypeptides for improved antibody production

用于改善抗体生产的工程化多肽

• 批准号: 7218917
• 负责人: Jun Joseph Wang
• 金额: $ 28.74万
• 依托单位: BIOO SCIENTIFIC CORPORATION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7218917
• 关键词: Affinity; Affinity Chromatography; Amino Acids; Antibodies; Antibody Formation; Antigens; Avidity; Bacteria; Basic Science; Binding; Biological; Biological Assay; Carrier Proteins; Cell Extracts; Cells; Characteristics; Chemicals; Chimeric Proteins; Chromatography; Column Chromatography; Communities; Complex; Detection; Development; Disease; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Escherichia coli; Goals; Haptens; Immunization; Immunoblotting; Keyhole Limpet Hemocyanin; Link; Market Research; Methods; Molecular Biology Techniques; Oryctolagus cuniculus; Peptide antibodies; Peptides; Performance; Phase; Phosphoamino Acids; Plasmid Cloning Vector; Procedures; Process; Production; Protein Engineering; Proteins; Proteome; Proteomics; Quaternary Protein Structure; Reagent; Research; Research Personnel; Sensitivity and Specificity; Series; Serum; Set protein; Side; Sodium Chloride; Specificity; Staging; System; Techniques; Technology; Temperature; Testing; Time; Work; analytical method; base; chemical synthesis; cost; expression cloning; expression vector; immunoaffinity chromatography; immunogenic; immunogenicity; improved; novel; peptide chemical synthesis; polyclonal antibody; polypeptide; recombinant peptide; response; scale up; tool; vector

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a new cost-effective method for the production of high-purity polyclonal antibodies. Polyclonal antibodies are important reagents used worldwide for antigen detection and purification in basic research as well as the detection of disease states. Increased availability of quality anti-peptide polyclonal antibodies would greatly benefit proteomic research. Unfortunately the production of polyclonal antibodies to peptide antigens currently requires the chemical synthesis and purification of peptide antigens, which is expensive and time-consuming. The proposed research will result in the development of new methods to produce purified polyclonal antibodies. These methods will allow the rapid production of recombinant peptide antigen/immunogen conjugates while eliminating both the need for chemical synthesis of peptide antigens as well as the chemical conjugation of peptide haptens to carrier immunogens such as KLH. The new production method will be amenable to high throughput production of high quality antibodies at a lower cost. Specific aims for Phase I: (1) Screen a series of proteins to identify new highly immunogenic proteins which can be recombinantly expressed in bacteria. (2) Develop a plasmid vector to allow the rapid cloning, expression and purification of peptide antigens fused to carrier immunogens identified in the screen in Aim 1. (3) Produce polyclonal antibodies to several peptides produced by immunizing rabbits with recombinant peptide-immunogen fusion proteins. Characterize the antibodies to identify the best overall carrier immunogen. In Phase I we will identify new immunogens which can be recombinantly-expressed in bacteria and then create an expression vector to allow the rapid cloning and expression of peptide antigens fused to the carrier immunogens. We will use protein-engineering techniques to improve the immunogenicity of the carrier even further. We will clone several peptide antigen sequences into the vector and express and purify the peptide-immunogen fusions from bacteria. We will then produce polyclonal antibodies in rabbits using the purified peptide fusion proteins. We will purify the antigen-specific antibodies from the serum using a two-step affinity-based procedure. Finally, we will evaluate the performance of each of the antibodies side-by-side and select the best performing carrier for further commercial development in Phase II. In Phase II, we will scale-up the most efficient system and broaden its utility for the production of a variety of diverse peptide antigens. We will also develop molecular biology techniques to incorporate modified amino acids (such as phosphoamino acids) into the peptide antigens expressed in bacteria. Anti-peptide polyclonal antibodies are important reagents for the detection and characterization of proteins in cells and other complex biological systems. The exquisite specificity of anti-peptide antibodies has been used to develop sensitive assays to examine characteristic changes in the proteome associated with diseased states. The ability of researchers to develop these assays is currently limited by the availability of high-quality antibodies. In Phase I we will develop a novel, commercially-viable method for high-throughput production of anti-peptide polyclonal antibodies. In Phase II, we will scale-up the most efficient system and broaden its utility for the production of a variety of diverse peptide antigens. We will also develop molecular biology techniques to incorporate modified amino acids (such as phosphoamino acids) into the peptide antigens expressed in bacteria. Our work will greatly improve the availability of low cost, high quality antibodies to the worldwide research community.

描述（由申请人提供）：该提案的总体目标是开发一种用于生产高纯度多克隆抗体的新方法。多克隆抗体是全球用于基础研究中抗原检测和纯化的重要试剂，以及疾病状态的检测。质量抗肽多克隆抗体的可用性增加将极大地受益于蛋白质组学研究。不幸的是，目前，对肽抗原的多克隆抗体的产生需要化学合成和纯化肽抗原，这是昂贵且耗时的。拟议的研究将导致开发新方法来产生纯化的多克隆抗体。这些方法将允许快速生产重组肽抗原/免疫原共轭物，同时消除了对肽抗原化学合成的需求，以及肽触发与KLH等载体免疫原子的化学结合。新的生产方法将以较低的成本适应高质量抗体的高吞吐量生产。第I期的具体目的：（1）筛选一系列蛋白质，以鉴定可以在细菌中重组表达的新型高度免疫原性蛋白。 （2）开发质粒载体，以允许在AIM 1中鉴定在筛网中鉴定到载体免疫原的肽抗原的快速克隆，表达和纯化1。表征抗体以识别最佳总体载体免疫原。在第一阶段，我们将确定可以在细菌中重组表达的新免疫原，然后创建一个表达载体，以允许融合到载体免疫原融合的肽抗原的快速克隆和表达。我们将使用蛋白质工程技术来进一步提高载体的免疫原性。我们将几个肽抗原序列克隆到载体中，并从细菌中表达和净化肽 - 免疫原融合。然后，我们将使用纯化的肽融合蛋白在兔子中产生多克隆抗体。我们将使用基于两步亲和力的过程从血清中纯化抗原特异性抗体。最后，我们将评估每种抗体并排的性能，并选择最佳性能的载体，以进一步的II期商业开发。在第二阶段，我们将扩大最有效的系统，并扩大其生产各种不同肽抗原的实用性。我们还将开发分子生物学技术，以将修饰的氨基酸（例如磷酸氨基酸）掺入细菌中表达的肽抗原中。抗肽多克隆抗体是细胞和其他复杂生物系统中蛋白质检测和表征的重要试剂。抗肽抗体的精美特异性已用于开发敏感测定法，以检查与患病状态相关的蛋白质组的特征变化。目前，研究人员开发这些测定的能力受高质量抗体的可用性限制。在第一阶段，我们将开发一种新型的，可商业可行的方法，用于高通量产生抗肽多克隆抗体。在第二阶段，我们将扩大最有效的系统，并扩大其生产各种不同肽抗原的实用性。我们还将开发分子生物学技术，以将修饰的氨基酸（例如磷酸氨基酸）掺入细菌中表达的肽抗原中。我们的工作将大大提高对全球研究社区的低成本，高质量抗体的可用性。