Toxin Gene Deleted C. perfringens as an Oral Delivery Vector

描述（由申请人提供）：C.产气荚膜梭菌肠毒素（cpe）启动子和该菌的生物学特性。A型产气荚膜杆菌使得将大量外源蛋白递送至末端回肠（胃肠道中粘膜免疫诱导位点的主要位置）成为可能，因此，可以用作抗原的一般口服递送载体。然而，这种递送载体存在两个潜在的问题。首先，C. A型产气荚膜杆菌产生两种细胞外毒素，α毒素（pic）和θ毒素（pfoA），其可引起气性坏疽，并且当用作疫苗载体时构成潜在危险。其次，C.从质粒表达外源抗原的产气荚膜杆菌具有与不稳定性和在环境中转移质粒编码的抗生素抗性基因相关的潜在问题。我们的假设是cpe阴性的C.产气荚膜杆菌具有从其染色体产生高水平的外源蛋白而无需抗生素选择的能力，将是将抗原递送至GALT中的免疫诱导PP的理想载体。本研究的总体目标是构建安全的重组C. A型产气荚膜杆菌，其不能产生毒素并稳定表达高水平的外源蛋白基因而不涉及抗生素抗性基因。这将通过a）敲除cpe阴性ATCC 3624 C的染色体pfoA基因来实现。B）通过target-tron方法将外源基因盒插入pfoA'突变体的染色体pic基因中以产生pIc'/pfoA'突变体，并在体外和体内测量外源蛋白从染色体DNA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、低成本和有效的口服递送载体。

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.