Toxin Gene Deleted C. perfringens as an Oral Delivery Vector

删除毒素基因的产气荚膜梭菌作为口服递送载体

• 批准号: 7187357
• 负责人: YUE CHEN
• 金额: $ 7.2万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7187357
• 关键词: Acids; American Type Culture Collection; Animals; Antibiotic Resistance; Antibiotics; Antigens; Autoimmune Responses; Bacterial Chromosomes; Bacterial Proteins; Biology; Characteristics; Chromosomes; Clostridium perfringens; DNA; Distal part of ileum; Dose; Enterotoxins; Environment; Enzymes; Exposure to; Gas Gangrene; Gastrointestinal tract structure; Gene Delivery; Gene Proteins; Genes; Goals; Gut associated lymphoid tissue; Human; Immune; Immune response; In Vitro; Infection; Inflammatory Bowel Diseases; Intestines; Invaded; Knock-out; Laboratories; Large Intestine; Location; Measures; Methods; Monitor; Mucosal Immune Responses; Mus; Open Reading Frames; Oral; Oral Administration; Organ; Organism; Plasmids; Proteins; Recombinants; SIV; Simian immunodeficiency virus Gag protein p27; Site; Small Intestines; Structure of aggregated lymphoid follicle of small intestine; System; Technology; Therapeutic Agents; Tissues; Toxin; Vaccination; alpha Toxin; base; commensal microbes; cost; cyclin-dependent kinase inhibitor 1B; extracellular; in vivo; interest; mutant; oral vaccine; p27 Cell Cycle Protein; p27 Enzyme Inhibitor; promoter; vector; vector vaccine

DESCRIPTION (provided by applicant): Unique characteristics of the C. perfringens enterotoxin (cpe) promoter and the biology of the C. perfringens type A has made it possible to deliver a large amount of foreign protein to the terminal ileum (a major location of the mucosal immune inductive sites in gastro-intestinal tract) and therefore, could be used as a general oral delivery vector of antigens. However, there are two potential problems with this delivery vector. First, C. perfringens type A produces two extracellular toxins, alpha toxin (pic) and theta toxin (pfoA) which could cause gas gangrene and pose a potential danger when used as a vaccine vector. Secondly, the C. perfringens expressed foreign antigen from a plasmid has potential problems related to instability and transferring a plasmid-encoded antibiotic resistant gene in the environment. Our hypothesis is that a pic /pfoA' mutant of cpe-negative C. perfringens, with the capacity to produce a high level of foreign proteins from its chromosome without antibiotic selection, will be an ideal vector to deliver the antigen to the immune inductive PPs in the GALT. The overall objective of this proposal is to construct a safe recombinant C. perfringens type A that is incapable of producing toxins and stably expresses high levels of foreign protein gene without involvement of antibiotic resistant gene. This will be accomplished by a) knocking out the chromosomal pfoA gene of a cpe negative ATCC 3624 C. perfringens type A using a mobile group II target-tron method; b) inserting a foreign gene cassette into the chromosomal pic gene of the pfoA' mutant by the target-tron methods to create a pIc'/pfoA' mutant and measure expression of the foreign protein from the chromosomal DNA in vitro and in vivo. Successful completion of this study will create a safe, low-cost and efficient oral delivery vector for vaccine and therapeutic agents.

描述(申请人提供)：产气荚膜梭菌肠毒素(CPE)启动子的独特特性和A型产气荚膜梭菌的生物学特性使其能够将大量外源蛋白输送到末端回肠(胃肠道粘膜免疫诱导部位的主要位置)，因此，可用作一般口服抗原输送载体。然而，这个传递载体有两个潜在的问题。首先，A型产气荚膜梭菌产生两种细胞外毒素，α毒素(PIC)和Theta毒素(PFOA)，它们可能导致气性坏疽，并在用作疫苗载体时构成潜在危险。其次，从质粒中表达外源抗原的产气荚膜梭菌存在潜在的问题，这与环境中的不稳定性和转移质粒编码的抗生素耐药基因有关。我们的假设是，CPE阴性产气荚膜梭菌的Pic/PfOA‘突变体能够在不选择抗生素的情况下从其染色体上产生高水平的外源蛋白，将是将抗原运送到GALT中免疫诱导的PPS的理想载体。本研究的总体目标是构建一株安全的、不产生毒素且稳定表达外源蛋白基因且不涉及抗生素耐药基因的重组A型产气荚膜梭菌。这将通过a)使用流动的第二组靶子方法敲除CPE阴性的ATCC 3624产气荚膜梭菌A型染色体的pFOA基因；b)通过靶子方法将外源基因盒插入到pFOA‘突变体的染色体基因中，创建Pic’/pFOA‘突变体，并在体外和体内检测染色体DNA中外源蛋白的表达。这项研究的成功完成将创造一种安全、低成本和高效的疫苗和治疗剂口服载体。