| Summary: | Lactococcuslactis(L. lactis) is a Gram-positive bacteria traditionally used in food
industry for flavouring and fermenting processes. In recent years, L. lactis was found
to be able to deliver plasmid DNA into eukaryotic cells and express heterologous
protein, which makes it being developed as safer alternative for vaccination. The
increasing demand of L. lactisbeyond application in food industry raises the need to
develop lactococcal bicistronic vector with target gene expression in the eukaryotic
system. The objective of this study is to construct a lactococcal vector containing
modified eukaryotic expression cassette, with internal ribosome entry site (IRES)
inserted between CMV promoter and Poly A signal to allow transcription and
translation of two proteins in a single cassette. IRES sequence was PCR-amplified
from pRetroX-IRES-ZsGreen1 vector, digested with EcoRV and XhoI, and ligated
into eukaryotic expression cassette from pcDNA 3.1 HisA. Constructed vector was
transformed into E. coli TOP 10 and the orientation of IRES fragment in the modified
eukaryotic expression cassette was confirmed by DNA sequencing. The modified
eukaryotic expression cassette was PCR-amplified from pcDNA3.1HisA/IRES before
sub-cloning into lactococcal vector pMG36e.pMG36eharbouringmodified eukaryotic
expression cassette was successfully transformed into E. coli TOP 10 but not L. lactis
NZ9000. Optimization of transformation protocol for L. lactis NZ9000 using
electroporation is needed to enhance transformation efficiency. Segragational
instability of constructed vector in E. coli host was detected, which may have been
caused by production of single-stranded intermediates and high-molecular weight
DNA due to rolling-cycle replication of the pMG36e vector.
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