The Reaserch About Production Liner DNA Via Large-Scale PCR And The Circulization Of Liner DNA Mediated By Recombinase

The site specific recombination system is that:a single polypeptide enzyme identify the specific DNA sequence,then make the sequence between the two sites to be exchanged、 recombinased、excised and reversed according to the relative direction of the two sites.The whole reaction could take place in vitro and need no additional energy or assistant factor.Based on the large-scale PCR technology that could produce liner DNA cheaply and efficiently,we try to establish a new way to get circular DNA:translate liner DNA into circular DNA by recombinase in vitro.It provide a theoretical basis to achieve high throughput production of circular DNA in cell-free system.The experimental results demonstrate that,we have successfully established a50mL PCR system via the DNA polymerases expressed in yeast which could harvest a product that have the similar concentration compared with regular PCR.In the while,the Cre recombinase has been expressed in E.coli.Preliminary results of the experiment show that it has the capability to circle liner DNA.These make it possible to produce circular DNA in vitro.Contrast with the traditional plasmid extraction methods,there’s no bacterial endotoxin and antibiotics pollution in our progress,the preparation period is shorter,the cost is lower,what’s more,It’s easily to remove expression irrelevant components. It inaugurate a new way to prepare circular DNA quickly with cell-free system.In the following reaserch,we attempt to prepare DNA vaccine with large-scale PCR. We can get a large number of "expression cassette"that encode some kind of antigenic protein,the product could directly use as liner DNA vaccine to immune the organisms after purification.The preliminary experiment indicate that,the liner DNA vaccine produced by large-scale PCR technology has certain protective immune responses against lethal infection with influenza virus,but the effect is far away with our expectation.As circular DNA is more stable then liner DNA,so we try to translate the liner DNA vaccine into circular DNA vaccine by recombinase and test will it get a better protective immune responses against lethal infection with influenza virus.This research bring a new strategy to product DNA vaccine massively and efficiency to respond rapidly to newly infectious diseases.