A class of polypeptides that promote pigs to produce African swine fever virus antigen-specific immune responses and applications thereof
An African swine fever virus and immune response technology, applied in the field of biomedicine, can solve problems that have not been reported yet, achieve the effects of no toxic side effects and biological safety hazards, promote immune response, and promote the proliferation of sensitized immune cells
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Embodiment 1
[0060] Solid phase synthesis and purity detection of embodiment 1 polypeptide
[0061] The polypeptide of the present invention is as follows:
[0062] SEQ ID NO.1: SMMMVFNQL, AP1;
[0063] SEQ ID NO.2: RGLMEITFML, AP2;
[0064] SEQ ID NO.3: MQWFMTMVI, AP3;
[0065] SEQ ID NO.4: YKLHGMRWF, AP4;
[0066] SEQ ID NO.5: HLLNFTLHCHA, AP5;
[0067] The above polypeptides were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The detection wavelength is 214nm. The purity of the final peptide purification product was >98%, and the structure was identified by ESI-MS. For the identification results, see Figure 1 to Figure 10 ,in figure 1 It is the identification chromatogram of AP1, and the peak time is 8.607min; figure 2 It is the mass spectrum of AP1, and the identified molecular weight is 1100.45g / mol, which is consistent with the theoretical value; image 3 It is the identification chromatogram of AP2, and the peak time is 15.534min; Figure 4 It is the mass spectr...
Embodiment 2
[0068] Embodiment 2 porcine lymphocyte proliferation experiment
[0069] 1. Pigs immunized with inactivated ASFV virus.
[0070] Five 90-day-old male Landrace pigs were immunized with ASFV epidemic strain inactivated virus (from the African swine fever regional laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) (10HID50), and a booster immunization was performed one month later, and the pigs were administered 7 days later. Euthanize and remove the spleen after dissection. Unimmunized healthy pigs were used as negative control group.
[0071] 2. Preparation, culture and proliferation detection of splenocytes.
[0072] 1) Aseptically treat the collected pig spleen with 75% alcohol, wash it three times with PBS, cut the spleen into small pieces, place them in folded sterile gauze (2 layers), and place them in 5 mL of serum-containing 1640 medium Grind the spleen in a plate.
[0073] 2) Then draw the liquid into a 15ml centrifuge tub...
Embodiment 3
[0082] Example 3 Detection of IFN-γ secretion by different subtypes of splenic lymphocytes
[0083] The pig immunization procedure and spleen cell isolation and culture are the same as in Example 2. After obtaining the dispersed splenocytes, prepare a single cell suspension with RPMI1640 complete medium at a concentration of 1×10 6 / ml. Inoculate into a 24-well plate, add 0.2 μg of the polypeptide (AP1-AP5) provided by the present invention to each well, and place in CO 2 Cultivate in the incubator for 60h. Collect the cells in each well, label the cells with porcine CD3, CD4, CD8 and IFN-γ specific antibodies respectively, then wash twice with PBS buffer containing 2% serum, and finally use the washing solution to disperse into a cell suspension, and use the flow Cytometer detection, respectively determine B lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, CD4 + CD8 + The levels of IFN-γ secreted by T lymphocytes and monocyte-macrophages.
[0084] For test resul...
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