Study On Genetic Engineering Of E Coli Chromosome Using The Recombineering System And A Novel Recombinant E.coli Vaccine Vector

As a new type of vaccine with gene insertion of specific antigen to bacterial chromosome, or plasmid vector which can present antigen against one or ones to lots of diseases, the recombinat live vaccine displays distinctive merit in inducing mucous immunity. Moreover, it can also induce cell immunity and humoral immunity. Currently, widely used as vaccine vector, attenuated bacteria ( e.g. salmonella ) has many disadvantages such as clinical side effect etc. So we need to study more safe and efficient live vaccine vector.Much more attention is paid to the study of normal E.coli as contagious disease vaccine vector or oncotherapy vector for the genetic background of E.coli is clearly known. In addition, it is safe and has less toxic effect. Its chromosome consists of 4.6×106 bases, which can be used as a vector of multivalent vaccine.To efficiently modify chromosome DNA of E.coli, express heterologous antigenic genes on chromosome and solve the problem of the disability of adhesion and invasion, this study focuses on the following four aspects: 1) to establish a technology platform of homologous recombination; 2) to screen efficient expression sites of exogenous gene on chromosome; 3) to construct a new E.coli recombinant strain with the ability of adhesion and invasion; 4) to evaluate the immunogenicity of the recombinant vaccine vector.Firstly, the recombinant engineering technology related vaccine preparation was set up, including: 1). the gene knock-out based on linear DNA target molecular in vivo; 2). the selection and counterselection technology of exogenous gene knock in definite site on chromosome; 3). the Gap-repair clone technology in vivo; 4). the recombination technology of single-chain DNA and the overlapping primer; 5). Set up the new selection and counterselection technology of neo-E. Secondly, to solve the problem of exogenous gene low-effective expression on chromosome, we transfer luciferase reporter gene into six interested gene expression sites in E.coli chromosome. The quantitative analysis results of exogenous gene expression displayed that it could efficiently express at lacZ site of lac operon after removing suppression gene lac I and the efficiency was obviously higher than multicopy plasmid. The fusion protein of foreign protein and outer membrane protein Lpp-ompA could be high-efficiently displayed on surface, which was the ideal way of antigen presentated by live bacterial vaccine. The stable expression of exogenous gene in E.coli chromosome had no effect on the bacterial growth and propagation. Meanwhile, for giving the E.coli with the ability of adhesion and invasion, we knocked invasion and adherence genes in outer membrane protein genes of E.coli DY330 chromosome, including a 71bp adherence...