Recombinant Borrel virus combined vaccine
A fowlpox virus and vaccine technology, applied in antiviral agents, antibody medical ingredients, medical preparations containing active ingredients, etc., can solve the problems of reducing vaccine immune protection and low attack protection
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Embodiment 1
[0056] Embodiment 1 Amplification, cloning and sequence analysis of chicken type II interferon gene
[0057] Culture of Chicken Spleen Lymphocytes
[0058] Aseptically take the chicken spleen according to the conventional method, put it in 5ml PBS, squeeze and collect the splenocytes, add the same amount of layered liquid of lymphocytes under the PBS liquid containing splenocytes, centrifuge at 1500rpm for 20min at room temperature, and collect at the interface After counting, add culture medium (DMEM containing 5% calf serum, glutamine containing 2mmol / L, HEPES 10mmol / L, concanavalin A (ConA) of 5μg / L) to adjust the splenocyte concentration to 10 7 Units / ml, placed at 41°C, 5% CO 2 Conditioned for 16h.
[0059] Extraction of total RNA containing interferon mRNA
[0060] Take 1ml of the chicken spleen lymphocytes cultured above (about 10 7 cells), placed in a 1.5ml centrifuge tube, centrifuged at 2500rpm for 10 minutes at 4°C, discarded the supernatant, added 1ml of TRIzo...
Embodiment 2
[0068] Example 2 Construction of FPV transfer vector (1175IFN) containing cIFN-II gene
[0069] The construction strategy of FPV transfer vector 1175IFN containing cIFN-II gene is shown in Figure 4.
[0070] After the above-mentioned steps in Example 1, in order to add the FPV early and late promoters in front of the cIFN-II gene sequence and smoothly connect to the FPV insertion vector 1175, the following four steps of conversion were performed.
[0071] First, pIFN was digested with SalI and HindIII to recover the cIFN-II gene fragment and inserted into the PE / L-containing pAF09 digested with SalI and HindIII to obtain a recombinant plasmid (AIFN). In AIFN, cIFN-II is located in the early and late stages of vaccinia virus Downstream of the promoter PE / L (Figure 5); followed by using the AIFN plasmid as a template to design a pair of primers
[0072] P3: 5'CCT GTC GAC AGC CAT TTA AGT ATC CCT AA 3’
[0073] P4: 5'GCG GTC GAC AAA AAA AAC TAT TAG CAA TTG CAT CTC CTC 3'),...
Embodiment 3
[0074] Embodiment 3 transfection and purification
[0075] The 1175IFN plasmid obtained in Example 2 was extracted in large quantities by alkaline lysis method, and the plasmid was purified by PEG method, OD 260 After determining the DNA content, fill it with ultra-pure water to make it 1 μg / ml for later use. DMEM containing 4% calf serum by 10 7cells / ml to prepare CEF, add 5ml of the above cell solution to each 100ml square bottle, and incubate at 37°C for 18h; then add wtFPV at an infection rate of 0.1 MOI, and adsorb at 37°C for 3h; wash the CEF monolayer twice with serum-free DMEM, Then add 2ml of serum-free DMEM for later use. Dilute 50 μg liposome transfection reagent (Dosper Lipofectin, Roche Company) and 10 μg 1175IFN to 50 μl in two 1.5ml finger tubes respectively, and the weight ratio of liposome and plasmid DNA is 5:1, and the above two tubes The substances were mixed, mixed gently, and allowed to stand at room temperature for 15 minutes. Then add the above-ment...
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