Polypeptide for promoting pig body to generate broad-spectrum immune response and application thereof
An immune response, pig body technology, applied in the field of biomedicine, can solve the problems of complex components, lack of immune enhancers, and high production costs, and achieve the effect of enhancing immune response and promoting antigen-specific immune response.
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Embodiment 1
[0062] Solid phase synthesis and purity detection of embodiment 1 polypeptide
[0063] The polypeptide of the present invention is as follows:
[0064] SEQ ID NO.1: KMRIEIFWEL;
[0065] SEQ ID NO.2: RSMMMVFNQL;
[0066] SEQ ID NO.3: YGIALQYVL;
[0067] SEQ ID NO.4: IFNFIENIWS;
[0068] SEQ ID NO.5: YTEMLALII;
[0069] The above polypeptides were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0070] 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 is the identification chromatogram of AP1, figure 2 It is the mass spectrum of AP1, and the identified molecular weight is 1364.85g / mol, which is consistent with the theoretical value; image 3 is the identification chromatogram of AP2, Figure 4 It is the mass spectrum of AP2, and the identified molecular weight is 1256.6g / mol, which is consistent wi...
Embodiment 2
[0071] Embodiment 2 porcine lymphocyte proliferation experiment
[0072] 1. Pigs immunized with inactivated ASFV virus.
[0073] 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.
[0074] 2. Preparation, culture and proliferation detection of splenocytes.
[0075] 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.
[0076] 2) Then draw the liquid into a 15ml centrifuge tub...
Embodiment 3
[0085] Example 3 Detection of IFN-γ secretion by different subtypes of splenic lymphocytes.
[0086] 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 provided by the present invention to each well, and culture in a CO2 incubator for 60 hours. 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, CD4 respectively + T lymphocytes and CD8 + Levels of IFN-γ secreted by T lymphocytes.
[0087] For test results, see Figure 13 ~ Figure 17 . in Figure 13 Promote the level of IFN-γ secreted by different ...
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