Application Of Homologous Recombination Techniques On The Construction Of Expression Vectors In Saccharomyces Cerevisiae

Saccharomyces cerevisiae is one of the most important heterologous expression systems. Yeast episomal plasmids containing complete sequence of 2μ, plasmid are now widely used in the expression vector construction of many genetic engineering drugs. However, the plasmid stability is not satisfying. These vectors contain a part of sequence from E.coli, including antibiotic resistance gene, which makes it unsuitable for the need of biological security. Another limitation is that the construction of this kind of vectors depends so much on restriction sites, while the prosess must be very complicated. To exert the power of the S. cerevisiae expression system, we should find out how to overcome these objections.S.cerevisiea is very efficient at homologous recombination. Observation shows that the amount of homology necessary to promote concise and efficient recombination in yeast is as small as 30~50bp. This dissertation is aiming to construct two kinds of yeast expression vectors by homologous recombination technique. The one is a set of highly stable pHR vectors, by which we can construct expression vectors without E.coli sequences. The other is vector pRSODF designed for hSOD-small peptide fusion expression.To construct the highly stable pHR expression vectors without E.coli sequence, we constructed the plasmid pHC11R based on pHC11. Then we choose hIFNa2b and HBV fusion antigen SA28 as target genes. The expression cassette of these two target genes were PCR amplified and two small periods of DNA were added on each end of the cassette for the sake of recombination. After getting rid of pHC11R's E.coli sequence, the remaining fragment was co-transformed into S. cerevisiae DCO4 with the amplified cassette. Then we got the genetic engineering strain of hIFNα2b and SA28: DCO4/pHC11R-IFNα2b and DCO4/pHC11R-SA28. The expression vector...