A two-color fluorescent protein-labeled duck plague virus and its construction method and application
A two-color fluorescence, protein labeling technology, applied in the field of molecular biology, can solve the problem of increasing the molecular weight of gC
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Embodiment 1
[0094] 1. Construction of pT-linker-mRFP-P1: using mRFP-F1 (gly) and mRFP-R1 as primer pairs (see Table 1), from the pLVX-mRFP-C1 (Clontech) plasmid template, PCR amplification prefixes are The mRFP gene of the flexible linker (GGGGSGGG) was inserted into the pMD19-T-simple vector (Takara), and colony PCR amplification was performed with the primer pair m13(+) and mRFP-R1 to screen the forward insertion clone pT-linker-mRFP; Enzyme cleavage site Pst I (CTGCAG);
[0095] Design primers kan_RFP-for(PstI) and kan_RFP-rev(PstI) (see Table 1), using pEP-kan-S as a template to amplify the fragment P1, the P1 fragment is introduced into the PstI-homology arm a-kan-Pst I; clone the fragment P1 into pT-linker-RFP using the PstI restriction site, and screen the forward insertion clone pT-linker-mRFP-P1 (forward insertion, this clone contains the linker-mRFP1-PstI-homology arm a- The Kan-PstI-mRFP2 fragment, mRFP1 and mRFP2 indicate that the mRFP is divided into two segments by the inse...
Embodiment 2
[0119] Rescue of recombinant virus
[0120] The DNAs of the BAC prepared in Example 1, Comparative Example 1 and Comparative Example 2 were extracted by alkaline lysis method, and the chicken embryo fibroblast CEFs were transfected according to the instructions of the Promega calcium phosphate transfection kit. 2 The culture was continued, and the virus was collected after 70-80% lesions appeared, and the rescued viruses were named rDEV-dGFP, rDEV-gCmRFP and rDEV-UL35CFP-gCmRFP, respectively.
[0121] Construction of recombinant BAC clones
[0122] The mutant clones and Kan intermediate DNA were extracted respectively, and identified by Xba I or Bgl II enzyme digestion ( image 3 ), electropherogram ( image 3 The middle right panel) and the results predicted by the reference sequence GenBank (EU082088.2) ( image 3 The middle left picture) is basically the same, and the PCR amplification fragment sequencing results of primer pairs dgfp-JD-F / R and CFP-UL35-JD-F / R are also c...
Embodiment 3
[0124] Determination of plaque size of recombinant virus
[0125] The rDEV-EF1, rDEV-dGFP, rDEV-gCmRFP and rDEV-UL35CFP-gCmRFP virus cryopreserved solutions were diluted with different dilutions and inoculated on a monolayer of CEFs in a 6-well plate. Cellulose-based DMEM medium at 37°C in CO 2 Incubate for 48h, take 100 plaque photos for each virus under a fluorescence microscope, use Image J software to measure the plaque area of different viruses, calculate the average value of each virus, and set the plaque area of rDEV-EF1 to 100 %, and other virus plaque areas are converted into percentages based on this standard. Statistical analysis was performed on these data with SPSS11.5 software.
[0126] Determination of plaque size of recombinant virus
[0127] After 48 hours of infection of CEFs cells with 4 strains of viruses, 100 photos of viral plaques were taken for each, and Image J software was used to measure the area of each plaque and calculate the average valu...
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