Hog fever virus ligand epitope polypeptide se242 and its application
A technology of SE242 and epitope polypeptide, which is applied to the swine fever virus ligand epitope polypeptide SE242 and its application field, can solve the problems of high synthesis cost, limited application and promotion, and no accurate positioning of E2 protein ligand epitope, etc. cost reduction effect
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Embodiment 1
[0021] In the present invention, multiple index measurements are carried out on the used CSF virus Shimen strain (cytotoxicity) to determine that the strain Shimen strain (cytotoxicity) CSFV can meet the use requirements of the present invention.
[0022] 1. ELISA titer determination of CSFV
[0023] Infect the cultured monolayer PK-15 cells with CSFV Shimen strain (cytotoxicity), harvest the virus after culturing at 37°C for 48-56 hours, freeze and thaw three times to break the cells to lyse the virus, centrifuge at 4°C, 3000rpm for 10min, Discard the precipitate, and the supernatant is the propagation virus solution. The harvested virus fluid was detected with a classical swine fever virus antigen detection kit (SERELISA HCV Ag Mono Indirect product). The virus liquid 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. with negative control OD 450 The ratio of the values is 2.2 times and the dilutio...
Embodiment 2
[0041] Embodiment 2, synthetic polypeptide
[0042] 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 SE242 is shown in figure 2 , the sequence is as follows:
[0043] SE242: SAGPVRKTSCTFNYAKTGKNKYYEPRDSYF (SEQ ID NO. 1)
Embodiment 3
[0044] Embodiment 3, SE242 and PK-15 cell combination and blocking test
[0045] 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.
[0046] Binding test of SE242 and PK-15 cells:
[0047] 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 reached 1.0×10 5 indivual. Add peptide SE242 (FITC-labeled) to the tube to make the final concentration of SE242 reach 0.2 mg / ml, and set FITC positive control at the same time, do three repetitions, keep on ice for 2 hours, wash with PBS 3 times, and centrifuge at 1000rpm for 5 minutes each time , after the last cent...
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