Recombinant Adenovirus with Epitope Insertions

In order to generate recombinant adenoviruses with the KWAS epitope genetically incorporated within Ad hexon HVR1 as well as His₆ incorporated within HVR2 or HVR5, the following was performed: for the HVR1 modification, the DNA sequence corresponding to a seven amino acid region of HIV gp41 was generated by GenScript and subcloned into the HVR1 region (the HIV sequence replaced amino acids 139 to 144) of the HVR2-His₆/pH5S or HVR5-His₆/pH5S plasmids [24] (link). In order to generate the control plasmid HVR1-His₆/pH5S, the DNA sequence corresponding to His₆ within the HVR1 (the His6 sequence replaced amino acids 139 to 144) was generated by GenScript and subcloned into the H5/pH5S plasmid [24] (link). The resulting plasmids HVR1-His₆/pH5S, HVR1-KWAS-HVR2-His₆/pH5S or HVR1-KWAS-HVR5-His₆/pH5S were digested with EcoRI and PmeI. These resulting fragments containing the homologous recombination regions and the hexon genes were purified, then recombined with a SwaI-digested Ad5 backbone vector that lacks the hexon gene, pAd5/ΔH5 [25] (link). These recombination reactions were performed in Escherichia coli BJ5183 (Stratagene, La Jolla, CA). The resultant clones were designated as Ad/H5-HVR1-His₆, Ad5/H5-HVR1-KWAS-HVR2-His₆ and Ad5/H5-HVR1-KWAS-HVR5-His₆. The control vector, Ad5/HVR2-MPER-L15ΔE1 was generated and characterized as previously described [5] (link). The Ad5 vector which is E1 deleted and contains green fluorescent protein and luciferase within the E1 region was generated and characterized as previously described [24] (link).

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Gu L., Li Z.C., Krendelchtchikov A., Krendelchtchikova V., Wu H, & Matthews Q.L. (2013). Using Multivalent Adenoviral Vectors for HIV Vaccination. PLoS ONE, 8(3), e60347.

Publication 2013

Adenoviruses Amino acids Backbone Clones Ecori Epitope Escherichia coli Gene Green fluorescent protein Hexon Homologous recombination Luciferase Plasmids Recombination Vector

Corresponding Organization :

Other organizations : University of Alabama at Birmingham, Tulane University

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Variable analysis

independent variables

Modification of the HVR1 region of the adenovirus hexon protein
Incorporation of the KWAS epitope within the HVR1 region
Incorporation of the His6 tag within the HVR2 or HVR5 region

dependent variables

Generation of recombinant adenoviruses with the KWAS epitope and His6 tag incorporated

control variables

Generation of the control plasmid HVR1-His6/pH5S, where the His6 sequence replaced amino acids 139 to 144 within the HVR1 region

controls

Positive control: Ad5/HVR2-MPER-L15ΔE1 vector, previously described
Positive control: Ad5 vector containing GFP and luciferase in the E1 region, previously described

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