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| 10mg |
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Purity: ≥98%
| Targets |
Immunology; class I MHC-restricted T-cell epitope
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| ln Vitro |
Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215-226) or class I (LLO 91-99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated delta aroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215-226 epitope, indicating that LLO 215-226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunization with the living, chromosomally integrated Salmonella construct carrying LLO 215-226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91-99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91-99 inserts: 91-99 (normal flagellin amino acids as flanking residues); KK91-99KK (Lys-Lys flanking residues); and AAA91-99AAA (Ala-Ala-Ala flanking residues)[1].
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| ln Vivo |
Spleen cells from mice immunized with the KK91–99KK flagellin chimeric Salmonella strain showed the greater CTL response indicating that Lys-Lys flanking residues are associated with enhanced immunogenicity, possibly by facilitating the proteolytic excision of the epitope in the endosomal compartment of antigen-presenting cells. A statistically significant decrease in the number of hepatic and/or splenic bacteria was achieved after Listeria challenge in mice immunized with Salmonella constructs expressing either MHC class II LLO 215–226 or class I LLO KK91–99KK flagellin chimera, showing that the immunogenicity of these constructs is associated with an epitope-specific increased resistance to infection[1].
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| Enzyme Assay |
DNA techiques[1]
Routine methods for recombinant DNA were as described by Maniatis et al. Plasmids were purified by the alkaline lysis method of Birnboim and Daly”. Bacterial strains were transformed by electroporation using the method developed by Dower et af.18. DNA sequencing was performed using the dideoxy chain- termination method of Sanger er ~1.‘~. For insertion of ofigonucleotides specifying T-cell epitopes in the cloned flagellin gene, the complementary oligonucleotides were annealed, kinased and blunt-end ligated to a phosphatase- treated EcoRV-digested DNA of pLS408’. Plasmid pLS408 has a 48 bp deletion (EcoRV fragment) in the central hy~rvariable region of the flagellin-coding sequence of the friC (HI-d) gene of S. muendren. The unique EcoRV site of pLS408 allows the blunt-end insertion of oligonucleotides. The ligated product was used to transform a flagellin-deficient E. co/i strain, CL447’, and transformants were selected on ampicillin plates. Recombinant plasmid carrying the chimeric flagellin gene, were sequenced using 18 bp primers located 78 bp upstream and 69 bp downstream from the insertion point. |
| Cell Assay |
The bacterial strains used in this report are shown in Table 1. Strains were grown at 37°C on Luria-Bertani broth (Difco Laboratories) supplemented with appropriate antibiotics. Minimal medium was made according to MilIer15. For whole bacterial @sates, log-phase growth cultures were pelleted by ce~t~fugation and washed twice with sterile PBS. Bacterial suspensions were lysed by sonication using Sonifier 250 ultrasonic cell disruptor for 1 min in bursts of about 10 s each. Protein concentration was determined using the Bradford protein assay reagent, For heat-killed preparations, Iog-phase growth cultures were centrifuged and washed twice in sterile PBS. Cell density was adjusted to 1 x IO9 ml-’ by determining the absorbance. Bacterial suspensions were heated at 80°C for 1 h and their viability was checked by plating aliquots onto LB-agar[1].
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| Animal Protocol |
Mice and immuni~rion protocol [1]
Balb/cByJ and C3Heb/Hej female mice, 6-8 weeks old, were obtained from Jackson Laboratories. Mice were immunized i.p. with a single dose of 1 x lo6 colony-forming units (c.f.u.) of recombinant Salmonella strains. Eight weeks after the injection, spleens were harvested from the immunized mice. Spleens from three to four mice in each group were pooled and used for IL-2 and CTL assays. In vivo protection assays[1] Balb/cByJ mice were immunized i.p. with 1 x lo6 c.f.u. of recombinant Scrlvrzonellrrstrains. Eight weeks thereafter, immunized mice were challenged i.p. with 2 x lo4 c.f.u. of L. monocytogenes (ATCC43251). The LD50 of this strain of L. nronoeytogenes for Balb/cByJ mice is < 5 x lo6 c.f.u./animal. Livers and spleens were harvested 72 h after infection and were homogenized in sterile PBS containing 0.5% NP40. Homogenates were serially diluted and plated on BHI-agar. Plates were incubated at 37’C for 24 h and the total number of colony-forming units was determined for each organ. The Mann-Whitney r test was employed in the analysis of results |
| References |
[1]. N K Verma, et al. Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella. Vaccine. 1995 Feb;13(2):142-50.
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| Molecular Formula |
C47H67N11O17
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|---|---|
| Molecular Weight |
1058.09839177132
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| Exact Mass |
1057.471
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| CAS # |
137856-41-8
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| PubChem CID |
25103379
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| Sequence |
Gly-Tyr-Lys-Asp-Gly-Asn-Glu-Tyr-Ile
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| SequenceShortening |
GYKDGNEYI
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| Appearance |
Typically exists as white to off-white solids at room temperature
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| LogP |
-7.5
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| Hydrogen Bond Donor Count |
16
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| Hydrogen Bond Acceptor Count |
19
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| Rotatable Bond Count |
34
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| Heavy Atom Count |
75
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| Complexity |
1980
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| Defined Atom Stereocenter Count |
8
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| SMILES |
CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)CN
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| InChi Key |
YDTMLXJRXDDANX-YTJLKFQJSA-N
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| InChi Code |
InChI=1S/C47H67N11O17/c1-3-24(2)40(47(74)75)58-46(73)32(19-26-9-13-28(60)14-10-26)56-43(70)30(15-16-38(64)65)55-45(72)33(20-35(50)61)53-37(63)23-51-41(68)34(21-39(66)67)57-42(69)29(6-4-5-17-48)54-44(71)31(52-36(62)22-49)18-25-7-11-27(59)12-8-25/h7-14,24,29-34,40,59-60H,3-6,15-23,48-49H2,1-2H3,(H2,50,61)(H,51,68)(H,52,62)(H,53,63)(H,54,71)(H,55,72)(H,56,70)(H,57,69)(H,58,73)(H,64,65)(H,66,67)(H,74,75)/t24-,29-,30-,31-,32-,33-,34-,40-/m0/s1
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| Chemical Name |
(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[(2-aminoacetyl)amino]-3-(4-hydroxyphenyl)propanoyl]amino]hexanoyl]amino]-3-carboxypropanoyl]amino]acetyl]amino]-4-oxobutanoyl]amino]-4-carboxybutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoic acid ;
glycyl-L-tyrosyl-L-lysyl-L-alpha-aspartyl-glycyl-L-asparagyl-L-alpha-glutamyl-L-tyrosyl-L-isoleucine
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| Synonyms |
LLO (91-99); 137856-41-8; AKOS-040756703; MS-31906; AKOS040756703; MS31906
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 100 mg/mL (94.51 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.9451 mL | 4.7255 mL | 9.4509 mL | |
| 5 mM | 0.1890 mL | 0.9451 mL | 1.8902 mL | |
| 10 mM | 0.0945 mL | 0.4725 mL | 0.9451 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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