Lactococcus lactis is being developed as a vaccine delivery system as it bears no
threat to animal and human health. It has been used for centuries in the fermentation
of foods and is generally recognised as safe. However, a safety factor pertaining to
the type of selectable marker present on the vector system poses to be of concern.
Chances of the transfer of antibiotic resistance genes, usually employed by vectors as
selectable markers, into the natural environment become a possible risk. Therefore,
there is a need for the development of ideal vectors without such selectable markers
(Dertzbaugh, 1998).
The activity of the xylanase gene of Bacillus coagulans ST -6 can be detected on
Remazol Brilliant Blue-Xylan (RBB-Xylan) as a clear halo zone against a dark blue
background. This characteristic allows xylanase to be used as a selectable
chromogenic marker on any vector system. On the other hand, the matrix or
membrane (M) protein of Newcastle disease virus (NDV) strain AF 2240 can be
useful as an antigen to generate antibody response towards NDV infection in
chickens.Both, the xylanase gene (0.8 kb) of B. coagulans ST-6 and the M gene (1.1 kb) of
NDV strain AF 2240 were cloned in lactococcal expression vector pMG36e and
transformed into Escherichia coli XLI-blue MRF'. The recombinant plasmids,
pMG36e-X and pMG36e-X-M were sub-cloned into L. lactis MG 1363 via
electroporation. The insertion and orientation of the xylanase gene was confmned
using restriction enzyme analysis and PCR amplification. Xylanase activity and
expression on RBB-Xylan agar plates further confmned its presence in the
recombinant plasmid pMG36e-X. In addition, the enzyme activity was also
quantitatively showed using the Somogyi-Nelson assay. The sequence of the M gene
obtained in clone pMG36e-X-M from L. lactis MG 1363 was found to be 99%
homologous to the established sequence (Jemain, 1999). Expression of the fusion M
protein was studied at the transcriptional leve1. RT -PCR was used to detect the
transcription of the gene using RNA of L. lactis MG1363 containing the recombinant
plasmid pMG36e-X-M as template. The size of the RT-PCR product correlated with
the size of the cloned M gene (1.1 kb). In addition, the RT-PCR product was
sequenced to confirm the presence of the M gene.
Based on the results obtained, recombinant plasmids pMG36e-X and pMG36e-X-M
were successfully constructed and introduced into E. coli XLI-blue-MRF' and L.
lactis MG1363. The recombinant DNA pMG36e-X is capable of expressing the
xylanase gene and can be further developed as a chromogenic selection marker for a
new and improved food-grade shuttle vector for E. coli and L. lactis. On the other
hand, expression of the fusion M gene was detected at transcriptional level in
recombinant clone pMG36e-X-M
