Method for enhancing immune response in treatment of infectious and malignant disease
An immune response and infectious technology, applied in allergic diseases, anti-infective drugs, vaccines, etc., can solve problems such as inhibiting anti-cancer immunity
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Embodiment 1
[0059] Example 1: Cloning of DNA vaccine constructs
[0060] In order to construct a DNA vaccine for CTLA-4, human or mouse CTLA-4 sequences were selected and combined with the transmembrane region of IL2 secretion signal sequence (IL2ss) and placental alkaline phosphatase (PLAP) at the N-terminus and C-terminus respectively The sequences were fused, and then constructed into a mammalian expression plasmid, pVAC-1. The resulting DNA sequences of pVAC1-IL2ss-hCTLA-4-PLAP (SEQ ID NO: 1) and pVAC1-IL2ss-mCTLA-4-PLAP (SEQ ID NO: 2) are listed in Figure 1a and Figure 1b middle. These DNA vaccines, such as human or mouse PD-1 or PD-L1 alone, and human or mouse fusion gene constructs composed of CTLA-4 and PD-1 and / or PD-L1, and in Those with or without the transmembrane region of PLAP at the C-terminus were constructed and listed below and Figure 10 middle:
[0061] (1) pVAC-1-IL2ss-hPD-L1 (containing the DNA sequence encoding human PD-L1);
[0062] (2) pVAC-1-IL2ss-hPD-1 (c...
Embodiment 2
[0071] Example 2: Construction and purification of recombinant polypeptides
[0072] In order to construct recombinant polypeptides of CTLA-4-PD-1 and CTLA-4-PD-L1, mouse CTLA-4 fused with PD1 or PD-L1 was constructed into the E. Coli expression vector, pET56. The resulting mCTLA-4-mPD-1-His was purified with a nickel-resin affinity column 6 and mCTLA-4-mPD-L1-His 6 The protein sequences are respectively SEQ ID NO:31 and SEQ ID NO:32.
Embodiment 3
[0073] Embodiment 3: preparation liposome and liposome / DNA complex
[0074]PC-PEG-PE liposomes were prepared as follows: 5.9 mg of PC (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine) was mixed with 14.6 mg of PEG (1,2-dipalmitoyl -sn-glycerol-3 phosphoethanolamine-N-polyethylene glycol-5000) (AvantiPolar Labs, Inc.) was dissolved in 2ml of solvent (90% chloroform, 10% MeOH) and 1ml of MeOH respectively, and placed in 500ml round bottom flask. Place the flask on a rotary evaporator at 55°C under vacuum until the liquid disappears. The flask was further subjected to three cycles of vacuum drying, room temperature for 30 minutes, and heating for 5 minutes. 6 mg polyethyleneimine (PE) (Sigma Aldrich) dissolved in 6 ml PBS was added to the rotating flask and five cycles of 10 minutes heating, 30 minutes room temperature were performed. The final solution was adjusted to 6 ml with distilled water and subjected to one freeze-thaw step (from -20°C to 4°C). The solution was filte...
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