EBIV nucleic acid composition and application thereof

A method for preparing a recombinant Ebinur Lake virus (EBIV) strain labeled with GFP fluorescence, comprising the following steps:

• Cloning genes shown in SEQ ID NO. 14-16, wherein primers used for amplifying the gene sequences were shown in SEQ ID NO. 14 are as shown in SEQ ID NO. 1 and 2;
• primers used for amplifying the genes shown in SEQ ID NO. 15 are shown in SEQ ID NO. 3 and 4;
• primers used for amplifying the genes shown in SEQ ID NO. 16 are shown in SEQ ID NO. 5 and 6;
• Ligating the genes shown in SEQ ID NO. 14-16 respectively to a linearized pLCK plasmid to obtain pLCK-EBIV-L, pLCK-EBIV-M, and pLCK-EBIV-S plasmids;
• Using pcDNA3.1-eGFP as a template, and eGFP-F and eGFP-R as primers to clone an eGFP fragment;
• Using pLCK-EBIV-S as a template, and CS-F and CS-R as primers to clone a linearized fragment of pLCK-EBIV-S;
• Ligating the eGFP fragment and the linearized fragment of pLCK-EBIV-S by homologous recombination to obtain a recombinant plasmid pLCK-EBIV-eGFP/S;
• wherein the eGFP fragment is ligated to pLCK-EBIV-S using P2A;
• the nucleic acid sequence of the P2A is shown in SEQ ID NO. 13;
• the nucleotide sequence of the eGFP fragment is shown in SEQ ID NO. 17;
• Mixing pLCK-EBIV-eGFP/S, pLCK-EBIV-M, and pLCK-EBIV-L in a mass ratio of 1:2:3, and co-transfect them into BSR-T7 cells and culture same, and harvest the transfected culture, wherein the culture contains the recombinant EBIV strain labeled with GFP fluorescence;
• wherein a method for constructing pLCK-EBIV-eGFP/S comprises:
• Cloning eGFP using primers eGFP-F and eGFP-R shown in SEQ ID NO. 9 and 10 from plasmid pcDNA3.1-eGFP to obtain the eGFP fragment shown in SEQ ID NO. 17;
• wherein the PCR reaction system of 50 μL:
• 2× Reaction Mix Buffer, 25 μL, 10 μM eGFP-F, 2 μL, 10 μM eGFP-R, 2 μL, Template DNA, 1 μL, and Nuclease-Free Water, 20 μL;
• the PCR amplification reaction:
• 98° C. for 10 s, 57° C. for 5 s, 68° C. for 30 s, 16° C. for 10 min;
• the number of cycles of 25;
• Cloning the pLCK-EBIV-S plasmid by using primers CS-F and CS-R shown in SEQ ID NO. 11 and 12 to obtain a linearized pLCK-EBIV-S fragment;
• wherein the PCR reaction system of 50 μL:
• 2× Reaction Mix Buffer, 25 μL, 10 μM CS-F, 2 μL, 10 μM CS-R, 2 μL, Template DNA, 1 μL, and Nuclease-Free Water, 20 μL;
• the PCR reaction condition:
• 98° C. for 10 s, 57° C. for 5 s, 68° C. for 30 s, 16° C. for 10 min;
• the number of cycles of 25;
• Ligating the eGFP fragment and the pLCK-EBIV-S linearized fragment by homologous recombination to obtain pLCK-EBIV-eGFP/S, wherein the reaction system for the homologous recombination of 20 μL:
• 5× Reaction Buffer, 4 μL, eGFP fragment, 30 ng, cS fragment, 60 ng, Enzyme, 2 μL, and Nuclease-Free Water, 33 μL;
• the homologous recombination reaction conditions:
• 37° C. for 30 min, and 16° C. for 10 min.