Cell strain for expressing Furin protein and application of cell strain in culture of avian infectious bronchitis virus
A bronchitis, cell line technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, viruses, etc., can solve problems such as changing viruses, infecting viruses, restricting virus infection and developing new vaccines with pathogenic mechanisms , to achieve the effect of simplifying the propagation process and enhancing the affinity
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Embodiment 1
[0088] The present embodiment carries out the acquisition and amplification of the chicken source Furin gene, specifically including:
[0089] Referring to the CDS sequence of chicken-derived Furin mRNA in NCBI, primers SEQ ID NO:2-3 were designed, and chicken-derived Furin gene SEQ ID NO:1 was amplified using DF-1 cell cDNA as a template. The reaction system is shown in Table 1, and the amplification system is shown in Table 1. Table 2.
[0090] Table 1
[0091] components volume 5× buffer 10 μL Upstream primer SEQ ID NO:2 (10μM) 2μL Downstream primer SEQ ID NO:3 (10μM) 2μL dNTPs (10mM) 1μL DNA polymerase 1μL cDNA template 1μL wxya 2 o
33μL total capacity 50μL
[0092] Table 2
[0093]
[0094] The amplified fragment was verified by agarose gel electrophoresis, and the electrophoresis results were as follows: figure 1 As shown, it can be seen from the electrophoresis results that the full length...
Embodiment 2
[0096] This example constructs a recombinant lentiviral vector Phage-ChF containing chicken-derived Furin gene, specifically including:
[0097] The Phage-puro vector was digested with Not I and Xba I in a water bath at 37°C for 4 hours, and the linearized plasmid fragment after digestion was recovered from the gel, and homologous recombination was performed with the chicken Furin PCR product using a homologous recombination kit. The homologous recombination system is shown in Table 3.
[0098] table 3
[0099] components volume Linearized Phage-puro vector 2μL Chicken Furin-PCR product 3μL 2×clone Express Mix 5μL total capacity 10 μL
[0100] The reaction conditions were to incubate at 50°C for 10 min, then place on ice immediately after cooling down to 4°C.
[0101] The recombinant product was transformed into Escherichia coli DH5α, cultured overnight in a 37°C incubator after plating, and positive colonies were selected the next...
Embodiment 3
[0104] In this embodiment, the packaging, transfection and concentration of recombinant lentivirus and negative control virus are carried out, specifically including:
[0105] 293T cells in good growth state were trypsinized, resuspended and inoculated in 10cm culture dish;
[0106] When the cell density reached 60%, the recombinant vector Phage-ChF, helper plasmids pMD2.G and psPAX2 were co-transfected into 293T cells, and the plasmids and transfection reagent Transport5 TM The usage ratio is 1:2;
[0107] Collect the cell supernatant after 60 hours, centrifuge at 3000g for 15 minutes at 4°C, filter through a 0.45μm filter, store the supernatant at -80°C, and prepare the recombinant lentivirus containing the chicken-derived Furin gene and the empty load of the negative control Lentivirus.
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