Recombinant vaccinia virus vector with D8L gene knocked out
A vaccinia virus vector and vaccinia virus technology, applied in the fields of molecular biology and immunology, can solve problems such as limiting the repeated or multiple application of vaccinia virus vectors, affecting the reinfection of the target recombinant vaccinia virus vector, and affecting the delivery effect of viral vector drugs , to achieve the effect of increasing the number of uses
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[0078] The present invention also provides a preparation method for constructing a D8L gene knockout recombinant vaccinia vector, comprising the following steps:
[0079] Step 1: designing primers to amplify the two homologous recombination arms of D8L by PCR respectively.
[0080] Step 2: Insert the two homologous recombination arms into the target shuttle vector to construct a shuttle vector containing the sequence of the D8L homologous recombination arm.
[0081] Step 3, transfecting the shuttle vector into vaccinia virus-infected cells.
[0082] Step 4, collecting the recombinant vaccinia virus vector from the cells, and performing single-spot purification on new cells.
[0083] Wherein, the primer is a primer sequence that can bind to both ends of D8L, preferably SEQ ID NO:3D8L-LC-F primer, SEQ ID NO:4D8L-LC-R primer, SEQ ID NO:5D8L-RC-F primer or SEQ ID NO : 6D8L-RC-R primer.
[0084] The shuttle vector is characterized by containing the D8L homologous recombination a...
Embodiment 1
[0098] Example 1 Construction of pΔD8L-GFP shuttle vector.
[0099] By designing primers (Table 1) on the Tiantan strain poxvirus TP5 clone genome (Genbank: KC207811) sequence, the genomic DNA of vaccinia virus wild-type rvv-WT (provided by Beijing Biological Products) (preparation method reference example 3) For template (respectively amplify D8L-L (SEQ ID NO: 1) and D8L-R (SEQ ID NO: 2) fragments, wherein, D8L-LC-F and D8L-LC-R primer pair is used for amplifying D8L- The L fragment, D8L-RC-F and D8L-RC-R primer pair were used to amplify the D8L-R fragment.
[0100] Table 1: Primers in Example 1
[0101]
[0102]
[0103] The first step is to construct the intermediate vector pΔD8L-L with D8L-L homologous recombination arm. First, use the pSC65-GFP vector transformed from the pSC65 shuttle vector (addgene, catalog number: 30327) (replacing the LacZ expressing galactosidase on the pSC65 vector with the EGFP gene expressing green fluorescent protein, provided by Suzhou ...
Embodiment 2
[0107] Example 2 Construction of recombinant vaccinia virus vector rvv-ΔD8L / G.
[0108] The recombinant vaccinia virus vector was obtained in 293T cells, and the specific method was as follows. On the first day, 293T cells were plated in 6-well cell culture plates (JET, TCP-010-006), 1×10 6 / well and incubated overnight at 37°C in a carbon dioxide cell incubator. On the second day, at 0.05MOI (ie 5×10 4 PFU (plaque forming unit) / well) was added to vaccinia virus wild strain rvv-WT (provided by Beijing Biological Products), and then placed in a 37°C carbon dioxide cell incubator and incubated for two hours, during which the shuttle vector / transfection reagent was prepared Complex. Wherein the shuttle carrier is the complex obtained from p obtained in Example 1. Among them, 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 s...
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