Dual-color fluorescent protein-labeled duck plague virus and construction method and application thereof
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. The construction of pT-linker-mRFP-P1: with mRFP-F1 (gly) and mRFP-R1 as a primer pair (see Table 1), from the pLVX-mRFP-C1 (Clontech) plasmid template, the PCR amplification prefix is The mRFP gene of the flexible linker (GGGGSGGG) was inserted into the pMD19-T-simple vector (Takara), and colony PCR amplification was performed with m13(+) and mRFP-R1 primers to screen the forward insertion clone pT-linker-mRFP; select a single gene on mRFP Restriction site Pst I (CTGCAG);
[0095] Design primers kan_RFP-for (PstI) and kan_RFP-rev (PstI) (see Table 1), with pEP-kan-S as template, for amplifying fragment P1, P1 fragment is introduced into PstI-homology arm a-kan-Pst I; Use the PstI restriction site to clone the fragment P1 into pT-linker-RFP, and screen the forward insertion clone pT-linker-mRFP-P1 (forward insertion, the clone contains linker-mRFP1-PstI-homology arm a- Kan-PstI-mRFP2 fragments, mRFP1 and mRFP2 indicate that mRFP is divided into two segments by the i...
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 respectively extracted by alkaline lysis, and chicken embryo fibroblast CEFs were transfected according to the instructions of the Promega calcium phosphate transfection kit. 2 The culture was continued, and the viruses were harvested after 70-80% of the lesions appeared, and the rescued viruses were named rDEV-dGFP, rDEV-gCmRFP and rDEV-UL35CFP-gCmRFP, respectively.
[0121] Construction of recombinant BAC clones
[0122] Each mutant clone and Kan intermediate DNA were extracted respectively, and identified by Xba I or Bgl II enzyme digestion ( image 3 ), electropherogram ( image 3 In b) and the result predicted by the reference sequence GenBank (EU082088.2) ( image 3 Middle a) is basically consistent, and the PCR amplified fragment sequencing results of the primer pair dgfp-JD-F / R and CFP-UL35-JD-F / R are also consistent with expect...
Embodiment 3
[0124] Determination of Plaque Size of Recombinant Viruses
[0125] Dilute the rDEV-EF1, rDEV-dGFP, rDEV-gCmRFP and rDEV-UL35CFP-gCmRFP virus frozen stocks with different dilutions, inoculate on the monolayer CEFs in a 6-well plate, and replace it with 1.5% formazan after 2 hours. Cellulose-based DMEM medium at 37°C in CO 2 After culturing in the incubator for 48 hours, take 100 plaque photos of each virus under a fluorescent 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 %, the plaque area of other viruses was converted into a percentage based on it. These data were statistically analyzed with SPSS11.5 software.
[0126] Determination results of plaque size of recombinant virus
[0127] After 48 hours of infection of CEFs cells by the 4 strains of viruses, 100 photos of virus plaques were taken, and the area of each plaque was measured by Image J...
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