Pseudorabies virus JS-2012 infectious clone plasmid and construction method and application
A pseudorabies virus, infectious cloning technology, applied in the field of bioengineering, can solve the problems that mutant strains cannot be completely protected, and the virulence of pseudorabies virus mutant strains is enhanced.
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Embodiment 1
[0059] The construction of embodiment 1 recombinant virus rJS2012-gG / loxp
[0060] In order to construct an infectious clone of pseudorabies virus JS-2012 strain, in this example, the loxp sequence and the marker gene EGFP were inserted into the JS-2012 genome by homologous recombination to obtain recombinant virus rJS2012-gG / EGFP. Then, the EGFP marker gene in rJS2012-gG / EGFP was deleted by Cre enzyme, and the recombinant virus rJS2012-gG / loxp carrying loxp was obtained.
[0061] The specific implementation process is as follows:
[0062] 1.1 Construction of transfer vector pTEGGF
[0063] First, pEGFP-C3 was digested with XhoI and BamHI, filled in with T4DNA polymerase, and the large fragment was recovered from the gel and ligated with T4DNA Ligase to obtain the plasmid pPEGFP-C3D that deleted the sequence of the multiple cloning site between XhoI and BamHI. Using the PRV JS-2012 genome as a template, primers PgGF / PgGR (Table 1) and PDgGF / PDgGR (Table 1) were used to ampli...
Embodiment 2
[0071] Embodiment 2 Construction of recombinant virus rJS2012-BAC
[0072] In this example, the loxp-Cre enzyme system and the loxp site in rJS2012-gG / loxp were used to recombine the artificial bacterial chromosome vector carrying the marker gene EGFP into rJS2012-gG / loxp to obtain the recombinant virus rJS2012-BAC. The specific implementation process is as follows:
[0073] 2.1 Construction of transfer vector pBeloBAC11-EGFP
[0074] Using pEGFP-C3D as template, use primers PEGFPF / PEGFPR (Table 1) to amplify the EGFP expression cassette, PCR reaction system: 94°C for 5min, 94°C for 30s, 57°C for 45s, 72°C for 2min, a total of 30 cycles; 72°C 10min. The results of electrophoresis of PCR products were as follows: Figure 6 ( Figure 6 Middle, 1: DNA Marker DL2000, 2: PCR amplified fragment), the target band of about 1600bp was amplified. The target DNA band and pBeloBAC11 were respectively digested with BamHI and HindIII, recovered by gel, and the EGFP expression cassette ...
Embodiment 3
[0077] The construction of embodiment 3PRV infectious bacteria artificial chromosome
[0078] In this example, the infectious plasmid pBAC-JS2012 was obtained by electrotransforming the rJS2012-BAC genome into Escherichia coli DH10B. The specific implementation process is as follows:
[0079] Infect vero cells with rJS2012-BAC, 16-21 hours after infection, use the Hirt method to extract the circular genome during rJS2012-BAC replication in the cells, and electroporate Escherichia coli DH10B. The electroporation conditions are as follows: 1mm electroporation cuvette, 1750V, 25μF , 200Ω, electric shock once. Add 0.8mL S.O.C culture solution immediately after electric shock, transfer the electrotransformed bacteria into a 2.0mL centrifuge tube, incubate at 37°C for 1h, centrifuge at 5000rmp for 2min, spread the precipitated bacteria on the LB plate containing chloramphenicol (25μg / mL), 37 Cultivate at ℃ for 24h. Bacterial colonies on the plate were picked, placed in LB liquid ...
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