A method for synthesizing the baculovirus genome and its application in the construction of recombinant baculovirus
A technology of baculovirus and synthetic method, which is applied in the direction of virus/bacteriophage, recombinant DNA technology, biochemical equipment and methods, etc., to achieve the effect of convenient construction, popularization and application, and convenient rescue
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[0031] The application of the above-mentioned synthetic method in the synthesis of recombinant baculovirus, which includes:
[0032] Using the baculovirus genome as a template, the 1-45 primer pairs in the primer combination were respectively subjected to PCR amplification reactions to obtain the 1-45 target genome fragments; the 1 target genome fragment was the product corresponding to the 1 primer pair, and The 2nd objective genome fragment is the product corresponding to the 2nd primer pair, and so on, the 45th objective genome fragment is the product corresponding to the 45th primer pair.
[0033] The amplified target fragments were recovered and purified to obtain 45 purified target segments; the PCR amplification reaction included using the baculovirus genomic DNA as a template to perform a PCR reaction;
[0034] The PCR reaction program is: 95°C, 5min; 95°C, 30s; 52-59°C, 30s; 68°C, 2min; 30 cycles; 68°C, 10min.
[0035] Through the PCR amplification reaction, a large ...
Embodiment 1
[0057] This embodiment provides a method for synthesizing the baculovirus genome. The synthesis method includes the use of a combination of primers and a shuttle vector. The amplification primer combination includes the 1-45th primer pair, and the base sequences of the 1-45th primer pair are respectively shown in SEQ ID No.1-90; the linear vector amplification primer combination includes the 46th-54th primer pair, and the 46th-45th primer pair The base sequences of the 54 primer pairs are respectively shown in SEQ ID No.91-108; the base sequence of the shuttle vector is shown in SEQ ID No.109.
[0058] The use of the primers provided in this example, the specific method is as follows:
[0059] 1.1 Genomic DNA, plasmid pGF and plasmid pGF-egfp of the baculovirus AcMNPV C6 strain were extracted.
[0060] 1.2 Perform PCR amplification reaction with primer pairs 1-45 respectively; the reaction system of PCR amplification reaction is: 5 μL of 10×KOD PCR buffer, 5 μL of dNTPs (2mM ...
Embodiment 2
[0076] This embodiment provides a method for synthesizing baculovirus and recombinant baculovirus, the method includes the primer combination provided in Example 1; the genome amplification primer combination and the linear vector amplification primer combination; the genome amplification primer combination includes the first- One or more of the 45 primer pairs, the base sequences of the 1-45 primer pairs are respectively shown in SEQ ID No.1-90; the linear vector amplification primer combination includes the 46-55 primer pairs, the 46- The base sequences of the 55 primer pairs are respectively shown in SEQ ID No.91-108.
[0077] For the use of the method, refer to the method provided in Example 1.
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