Toxin Gene Deleted C. perfringens as an Oral Delivery Vector

描述（由申请人提供）：A型产气荚膜荚膜菌肠毒素启动子（cpe）的独特特性和A型产气荚膜荚膜菌的生物学特性使其能够将大量外源蛋白递送到回肠末端（胃肠道粘膜免疫诱导部位的主要位置），因此可作为抗原的一般口服递送载体。然而，这种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α毒素（pfoA），这两种毒素可能导致气性坏疽，并在用作疫苗载体时构成潜在危险。其次,外国抗原表达的c . perfringens从质粒不稳定和潜在问题相关传输plasmid-encoded抗生素耐药基因在环境中。我们的假设是，一个pic /pfoA'突变的cpe阴性产气荚膜梭菌，具有从其染色体产生高水平的外源蛋白的能力，无需抗生素选择，将是一个理想的载体，将抗原传递给GALT中的免疫诱导PPs。本研究的总体目标是构建一种安全的重组产气荚膜荚膜菌a型，该a型不产生毒素，稳定表达高水平的外源蛋白基因，不涉及抗生素耐药基因。这将通过a)使用移动II组靶-tron方法敲除cpe阴性ATCC 3624 C.产气荚膜菌a型的染色体pfoA基因来实现；b)将外源基因盒插入pfoA‘突变体的染色体pic基因中，通过靶-tron方法创建pic ’/pfoA'突变体，并在体外和体内测量来自染色体DNA的外源蛋白的表达。这项研究的成功完成将为疫苗和治疗剂创造一种安全、低成本和高效的口服递送载体。

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.