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Synthetic Gene 112


 
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Field NameNatural GeneSynthetic Gene
SGDB Gene ID101112
GenBank AccessionAY750900
GenBank GI53987910
Gene NameLLO (listeriolysin O bps. 91-99)LLO (mammalianized listeriolysin O bps. 91-99)
Gene Length (bp)5757
SpeciesListeria monocytogenesMus musculus (spleen and liver cells)
StrainsEGDBALB/c
CDSatgggttacaaagatggaaatgaatatattaaactcgaggaagacgccaaaaacataatgggctacaaggacggcaacgagtacatcaagctcgaggaagacgccaaaaacata
5' End
3' Endtagtag
Notes
Expression Vectorp91wt (contains a Luciferase fusion)p91mam (contains a Luciferase fusion)
Assay MethodsFlow cytometry, RNA gel, Luciferase activity relative to cotransfected CMV promoted β-gal, bacterial titer of organsFlow cytometry, RNA gel, Luciferase activity relative to cotransfected CMV promoted β-gal, bacterial titer of organs
ResultsThe mice were infected with L. monocytes after being treated with the plasmid. No cytotoxic T cell (CTL) response to LLO 91-99 coated and noncoated J774 cells. Interferon-gamma levels were similar to background level; there was no upregulation of immune rThe mice were infected with L. monocytes after being treated with the plasmid. Cytotoxic T cells lysed LLO 91-99 coated J774 cells, but did not lyse noncoated bacterial cells. Interferon-gamma levels reached 41,522 pg/ml. The number of infectious bacteri
Protein Functioncytotoxin (sulfhydryl-activated) which permits escape from phagocytic vesicles and entry into the cytosol
Recoding PurposeTo improve expression
Synthesized ByBio-Synthesis (Lewisville, TX)
Recoding MethodComparison of RSCU values from 2,939,102 Mus musculus and 28,162 codons from L. monocytogenes
indicated poor translation efficiency. The recoding pushed all used codon’s RSCU values in mice
above 1.100.
Publication Author(s)Uchijima, M.; Yoshida, A.; Nagata, T.; Koide, Y.
Corresponding AuthorYukio Koide
Corresponding AddressDepartment of Microbiology and Immunology, Hamamatsu University School of Medicine, Japan.
Publication Year1998
Publication TitleOptimization of codon usage of plasmid DNA vaccine is required for the effective MHC class I-restricted T cell responses against an intracellular bacterium
AbstractIn an attempt to study codon usage effects of DNA vaccines on the induction of MHC class I-restricted T cell responses against an intracellular bacterium, Listeria monocytogenes, we designed two plasmid DNA vaccines encoding an H-2Kd-restricted epitope of listeriolysin O (LLO) of L. monocytogenes, LLO 91-99. One DNA vaccine, p91wt, carries the wild-type DNA sequence encoding LLO 91-99, and the other one, p91mam, possesses the altered DNA sequence in which the codon usage was optimized for murine system. Our read-through analyses with LLO 91-99/luciferase fusion genes confirmed that the optimized 91mam DNA sequence showed extremely higher translation efficiency than the wild-type sequence in murine cells. Consistent with this, i.m. injections of p91mam, but not of p91wt, into BALB/c mice were capable of inducing specific CTL- and IFN-gamma-producing CD8+ T cells able to confer partial protection against listerial challenge. Taken together, these observations suggest that optimization of codon should be taken into consideration in the construction of DNA vaccines against nonviral pathogens.
JournalJ Immunol. 161(10): 5594-9.
SummaryThe authors wished to develop a DNA vaccine for mammals against the Listeria monocytogenes. They exploited the LLO (Listeriolysin O) 91-99 bp region which codes for the antibody binding site. Normally, a L. monocytogenes induces both MHC class I-restricted and MHC class II-restricted CD4+ T cell responses. The intracellular bacterium escapes from its phagocytic vesicle when it secretes listeriolysin O. However, if the infected T cell contains an epitope of LLO, then it is protected from the escaping bacteria. To integrate the epitope of LLO into T cells, and thus synthesize a vaccine, the authors inject a plasmid (p91wt) including the nucleotide sequence encoding LLO base pairs 91-99, hoping for the expression of an effective epitope. The authors wanted to then be able to illicit cytotoxic T cell (CTL) response from mouse cells upon infection by L. monocytogenes. However, the wild-type gene was too poorly translated and unable to produce the response due to the murine environment. These mice fared no mice than mice that received the control treatment. The plasmid (p91mam) including the recoded gene, however, was able to immunize the mice. Upon infection with bacteria expressing the LLO 91-99 on their coats, mice that received p91mam were able to upregulate immune response, producing far more interferon-gamma and interleukin-4 than the mice which received p91wt. Further analysis showed mice inoculated with p91mam were able to bring the number of infectious bacterial cells down to levels 10x to 100x lower than mice inoculated with p91wt. Overall, the results indicate that the p91wt had very little effect, almost the same as a control group receiving no plasmid. The p91mam group, however, gained immunity to LLO 91-99 coated L. monocytogenes.
Comments
Discussion
PubMed ID9820537
Submitter NameZheng, Yuanpu
Submitter AddressUMBC
Entry ConfirmationNo
 
 

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