Construction and application of Escherichia Coli-Riemerella anatipestifer efficient shuttle plasmid pFY02 / 生物工程学报

Riemerella anatipestifer is a pathogen that mainly infects ducks, gooses, turkeys and other birds, causing septicemia and serositis. At present, the function of R. anatipestifer genes are studied by gene deletion and complementation. However, the shuttle plasmid pLMF03 used at present is inefficient for conjugation. Moreover, less restriction enzyme site can be used for cloning. It is not able to use for all the genes complementation. To solve this disadvantage, the conjugative transfer site, R. anatipestifer replication initiation gene, high expression promoter and a number of enzyme cutting sites were cloned into the plasmid pPM5, to generate the new shuttle plasmid pFY02. The shuttle plasmid pFY02 was stable in R. anatipestifer and had a high conjugative transfer efficiency. The R. anatipestifer tonB2 mutant strain could be complemented by shuttle plasmid pFY02 expressing tonB2, indicating that the shuttle plasmid can be used to the complementation of R. anatipestifer. Taken together, the new shuttle plasmid pFY02 constructed in this study replenishes the genetic tool for complementation.

Construction and identification Bifidobacterium as a delivery system of IL -10 / 中国基层医药

Objective To construct and identify a novel IL-10 delivery system by transforming a hIL-10-containing plasmid into B.longum (BL -hIL -10).Methods A plasmid vector pBADs -GFP was selected which had been built by previous test and biosynthetic hIL-10 plasmid,through double enzyme digestion and enzyme reaction,to construct and identify PBADs-hIL-10 shuttle plasmid,then to synthesis BL-hIL-10.hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after 0.2% L-arabinose induction in vitro as examined by Western blot,enzyme-linked immunosorbent assay (ELISA)and RT-PCR;Culture supernatants and bacterium pellets were collected after continuous culture for 12,24 and 36h,respectively.hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after 0.2% L-arabinose induction in vitro as examined by Western blot,enzyme -linked immunosorbent assay (ELISA)and RT -PCR;Culture supernatants and bacterium pellets were collected after continuous culture for 12,24 and 36h,respectively.Results The BL-hIL-10 bacterial strain that can stably express hIL-10 factor was successfully screened out,and the levels of hIL-10 in both superna-tant and cell pellet were similarly reached maximum at 24h of culture.Conclusion BL-hIL-10 as a novel oral hIL-10 delivery system has been successfully established,which established a basis for the treatment of IBS with transgenic Bifidobacterium.

A Convenient Method to Select DNA Fragments of Cephalosporium acremonium with Promoter Function / 微生物学通报

A promoter-trap vector pGBT14 for selecting promoters of fungus gene was constructed with E. coli-yeast shuttling plasmid pGBT9. Using this vector, a0. 5-2. 0kb chromosomal DNA library of Cepholosporium acremonium was constructed, and twenty four DNA fragments with promoter function in Saccharomyces oerevisiae Y153 were selected from this DNA library. And the promoter function of these DNA fragments was analyzed.