Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody in vivo
A vaccinia virus vector and vaccinia virus technology, applied in the fields of molecular biology and immunology, can solve the problems of restricting repeated or multiple applications of vaccinia virus vectors, affecting the drug delivery effect of the viral vector, affecting the reinfection of the target recombinant vaccinia virus vector, etc. , to achieve the effect of increasing the number of uses
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[0077] The present invention also provides a preparation method for constructing A27L gene knockout recombinant vaccinia virus vector, comprising the following steps:
[0078] Step 1: Design primers to respectively amplify the two homologous recombination arms of A27L by PCR.
[0079] Step 2: Insert the two homologous recombination arms into the target shuttle vector to construct a shuttle vector containing the A27L homologous recombination arm sequence.
[0080] Step 3: Transfect the shuttle vector into the vaccinia virus-infected cells.
[0081] Step 4. Collect the recombinant vaccinia virus vector from the cells and perform single-spot purification on new cells.
[0082] Wherein, the primer is a primer sequence that can bind to both ends of A27L, preferably SEQ ID NO:3A27L-LC-F primer, SEQ ID NO:4A27L-LC-R primer, SEQ ID NO:5A27L-RC-F primer or SEQ ID NO : 6A27L-RC-R primer.
[0083] The shuttle vector is characterized by containing the A27L homologous recombination arm ...
Embodiment 1
[0098] Example 1pΔA27L-GFP shuttle vector construction.
[0099] By designing primers (Table 1) on the sequence of Tiantan strain poxvirus TP5 cloned genome (Genbank: KC207811), the genomic DNA of vaccinia virus wild-type rvv-WT (provided by Beijing Biological Products) (refer to Example 3 for preparation method) is the template (respectively amplified A27L-L (SEQ ID NO: 1) and A27L-R (SEQ ID NO: 2) fragments, wherein, A27L-LC-F and A27L-LC-R primer pairs are used to amplify A27L- The L fragment, A27L-RC-F and A27L-RC-R primer pairs were used to amplify the A27L-R fragment.
[0100] Table 1: Primers in Example 1
[0101]
[0102]
[0103] The first step is to construct the intermediate vector pΔA27L-L-GFP with the A27L-L homologous recombination arm. First, the pSC65-GFP vector (the LacZ expressing galactosidase on the pSC65 vector was replaced with the EGFP gene expressing green fluorescent protein), which was transformed from the pSC65 shuttle vector (addgene, catalo...
Embodiment 2
[0107] Example 2 Construction of recombinant vaccinia virus vector rvv-ΔA27L / G.
[0108] The recombinant vaccinia virus vector was obtained in 293T cells, and the specific method was as follows. On day 1, 293T cells were plated in 6-well cell culture plates (JET, TCP-010-006), 1×10 6 / well, incubate overnight at 37°C in a carbon dioxide cell incubator. The next day, at 0.05MOI (ie 5×10 4 PFU (plaque forming unit) / well) was added to the vaccinia virus wild strain rvv-WT (provided by Beijing Biological Products), and then placed in a 37°C carbon dioxide cell incubator for two hours, during which the shuttle vector / transfection reagent was prepared Complex. The shuttle vector is pΔA27L-GFP obtained in Example 1, the transfection reagent is Turbofect (Thermo Fisher Scientific, R0531), and the transfection dose and compounding method can be found in the instructions of the transfection reagent. After the complex system was completed, the 293T cell supernatant was replaced with ...
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