Hog fever virus ligand epitope polypeptide se241 and its application
A technology of SE241 and epitope polypeptides, which is applied to the ligand epitope polypeptide SE241 of swine fever virus and its application fields, can solve the problems of high synthesis cost, precise positioning of E2 protein ligand epitopes that have not yet been seen, and restrictions on application and promotion. The effect of cost reduction
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Embodiment 1
[0020] In the present invention, multiple indicators are determined for the Shimen strain (cytotoxic) of the swine fever virus used to determine that the Shimen strain (cytotoxic) CSFV can meet the use requirements of the present invention.
[0021] 1. ELISA titer determination of CSFV
[0022] Infect the cultured monolayer PK-15 cells with the Shimen strain of swine fever virus (cytotoxicity), harvest the virus after culturing at 37°C for 48-56h, and then freeze and thaw 3 times to break the cells to lyse the virus. Centrifuge at 4°C and 3000rpm for 10min. The precipitate was discarded, and the supernatant was the virus propagating solution. The harvested virus liquid was detected with a swine fever virus antigen detection kit (SERELISA HCV Ag Mono Indirect product). The virus solution was diluted 1:50, 1:100, 1:200, 1:400 times, and each dilution was repeated 6 times. The test results are shown in Table 1. to the OD of the negative control 450 The ratio of the values is...
Embodiment 2
[0040] Embodiment 2, synthetic polypeptide
[0041] Studies have shown that the peptide SE24 (amino acid sequence: CVHASDERLGPMPCRPKEIGSSAGPVRKTSCTFNYAKTGKNKYYEPRDSYF) can effectively bind to PK-15 cells, and can effectively inhibit CSFV from infecting PK-15 cells, and CSFV can block SE24 from binding to PK-15 cells, so it is confirmed that the peptide SE24 is CSFV E2 protein ligand epitope polypeptide. In order to further accurately locate the ligand epitope information on the peptide SE24, a short peptide located on the peptide SE24 was designed and synthesized. The LC-MS / MS spectrum of the synthetic peptide SE241 is shown in figure 2 , the sequence is as follows:
[0042] SE241: VHASDERLGPMPCRPKEIGSSAGPVRKTSC (SEQ ID NO: 1)
Embodiment 3
[0043] Embodiment 3, SE241 and PK-15 cell combination and blocking test
[0044] Take well-growing PK15 cells, wash them with PBS for 3 times, digest the cells with 1% trypsin for about 1 min, discard the digestion solution, add DMEM containing 10% fetal bovine serum to suspend, centrifuge at 1000rpm for 5 min, and resuspend with an appropriate amount of PBS. Suspend PK15 cells, count and adjust the cell concentration to 1.0×10 6 pcs / ml, spare.
[0045] Binding test of SE241 and PK-15 cells:
[0046] The above PK15 cell suspension (cell concentration is 1.0×10 6 cells / ml), mixed evenly and placed in EP tubes, 100 μl in each tube, that is, the number of cells in each tube reached 1.0×10 5 indivual. Add peptide SE241 (FITC-labeled) to the tube to make the final concentration of SE241 reach 0.2 mg / ml, and set FITC positive control at the same time, do three repetitions, keep it on ice for 2 hours, wash with PBS for 3 times, and centrifuge at 1000rpm for 5min each time , afte...
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