A cell line capable of inducible and stable expression of Peste des petits ruminants virus p protein and use thereof
A kind of PPR, cell line technology, applied in the field of cell biotechnology and animal health, can solve the problem of difficult to achieve stable and high-efficiency expression of P protein inducible
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[0032] More specifically, the vaccine is a vaccine against Peste des petits ruminants. Wherein, the preparation method of the vaccine may include: using the cell line as described above to prepare purified PPR virus P protein, and then mixing the purified PPR virus P protein with an immune adjuvant.
[0033] On the other hand, the present disclosure also provides the use of the above-mentioned cell line in the preparation of a test kit.
[0034] Specifically, the test kit is a test kit for testing whether the animal to be tested carries antibodies against PPR virus. More specifically, the test kit is a test kit for testing whether the animal to be tested carries an antibody against the P protein of PPR virus. Animals to be tested may be detected by the above test kits if they have been exposed to, have been infected with, or have been vaccinated against PPR.
[0035] Wherein, as mentioned above, the nucleic acid sequence of the gene encoding the P protein of Peste des petits...
Embodiment 1
[0038] This example is used to describe in detail: the establishment of doxycycline-inducible 293TREX-PPRV-P cell line.
[0039] Materials used in the experiment: pCDNA4 / TO plasmid, pCDNA6 / TR plasmid, TREx-293 cells.
[0040] The specific steps of the experiment: The P gene coding sequence of PPRV is amplified and inserted into the restriction enzyme cleavage site of the pcDNA4to vector as shown in Figure (1A) to generate the pCDNA4-To-PPRV-P recombinant expression vector, and the sequence is verified by sequencing. The pCDNA4-To-PPRV-P plasmid was transferred into TREx-293 cells that had been stably transformed into the pcNDA6 / TR plasmid, as shown in Figure (1B), and 10 μg / mL blasticidin and 300 μg / mL Blastidin were added. Zeocin was used to screen out the surviving cells of the cell clones stably transferred into the pcNDA6 / TR and pCDNA4-To-PPRV-P plasmids, and the cells were continuously proliferated and developed into cell lines, which were cultured in 10% fetal bovine ser...
Embodiment 2
[0042] This example is used to describe in detail: the expression of P protein in 293TREX-PPRV-P cell line induced by doxycycline.
[0043] Cells used in the experiment: 293TREX-PPRV-P cell line.
[0044] The specific steps of the experiment: inoculate the 293TREX-PPRV-P cell line on a six-well plate and continue to culture for 24 hours, add (10ng / mL) doxycycline to the medium of the treatment group, and continue to culture for 24 hours (or After 48 and 72 hours), total protein and total RNA were extracted; the above protein samples were subjected to immunoblotting analysis. Compared with the control group without doxycycline, doxycycline could significantly induce PPRV-P in 293TREX-PPRV-P cell lines. expression of P protein (see Figure 2A ), and the expression of P protein was induced time-dependent (see Figure 2B ) model and doxycycline dose-dependent model (see Figure 2C ); reverse transcription PCR and fluorescence relative quantitative PCR analysis of the above tota...
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