[Show abstract][Hide abstract] ABSTRACT: Even though mRNA quantification provides significant information for biological analysis, current methods such as Northern blot analysis and real-time PCR are known to be laborious and lacking in precision. In this study, we demonstrate a new precise mRNA quantification method using CE based on SSCP (CE-SSCP) coupled with reverse transcription. mRNA samples could be simply analyzed for the quantification directly with reverse transcript obtained from a single reaction. This helps to avoid considerable errors generated by a series of the tedious manual steps. Also, unlike real-time PCR, reverse transcripts can be directly quantified by CE-SSCP in this method without further data estimation. Reproducibility and accuracy of CE-SSCP for mRNA quantification was examined using enhanced green fluorescent protein (eGFP) mRNA transcribed in vitro. Specific reverse transcription primer was determined for the accurate quantification of eGFP mRNA from total RNA obtained from the recombinant Escherichia coli. Using elongation factor Tu mRNA as an internal standard, it was shown that sample-to-sample variation could be minimized. Expression kinetics at both mRNA level and protein level was studied and the potential of CE-SSCP in expression analysis was demonstrated by comparison with the eGFP activity assay.
[Show abstract][Hide abstract] ABSTRACT: A DNA vaccine methodology using eukaryote expression vectors to produce immunizing proteins in the vaccinated hosts is a novel
approach to the development of vaccine and immuno-therapeutics, and it has achieved considerable success over several infectious
diseases and various cancers. To further enhance its efficiency, attempts were made to develop novel plasmid vectors containing
multiple immunostimulatory CpG motifs, for rapid and strong immune response. First, a 2.9 kb compact plasmid vector (pVAC),
containing CMV promoter, polycloning site, BGH poly(A) terminator, ampicillin resistance gene and pBR322 origin was constructed.
A pVAC-hEPO was also constructed, which contained a human erythropoietin gene, for evaluating the transfection efficiency
of naked plasmid DNA bothin vitro andin vivo. To examine the adjuvant effect of multi-CpG motifs on naked plasmid DNA, 22 and 44 enriched and unmethylated CpG motifs
were introduced into pVAC to generate pVAC-ISS1 and pVAC-ISS2, respectively. 100 μg of pSecTagB, pVAC, pVAC-ISS1 or pVAC-ISS2
were each injected intramuscularly into the tibilias anterior muscle of Balb/c mice. The level of interleukin-6 induced in
the mice injected with pVAC-ISS1 and pVAC-ISS2 were significantly elevated, after 12 hours, which were almost 2 and 2.5 times
higher than that in the mice injected with pSecTagB, respectively. These results suggest that DNA vaccine plasmids with enriched
CpG motifs can induce rapid secretion of interleukin-6 by lymphocytes. In conclusion, these vectors can contribute to the
development of adjuvant-free DNA vaccinations against infectious diseases and various cancers.
[Show abstract][Hide abstract] ABSTRACT: An efficient cell-free translation system was developed by removal of phosphatase localized in the periplasmic space, which hampers the translation reaction by hydrolyzing ATP. S30 extract was prepared from the spheroplast of Escherichia coli, and as much as 40% of ATP-hydrolysis activity of phosphatases could be easily removed by the spheroplast formation. The reaction period of translation using phosphatase-removed S30 extract could be prolonged and protein synthesis was enhanced by more than 30%.