Recombinant adenovirus with anti-tumor immune function as well as preparation method and application of recombinant adenovirus
A recombinant adenovirus, anti-tumor immunity technology, applied in anti-tumor drugs, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as drug resistance of monoclonal antibodies, and achieve the effect of immunotherapy
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Embodiment 1
[0028] Example 1 Screening of Polypeptide PP4
[0029] 1) Analyze the protein sequence of the avirulent mutant of diphtheria toxin, predict the CD4+ T cell epitope of CRM197 by NetMHCⅡpan4.0Server software, and screen out the peptides with medium and strong affinity, and obtain 9 peptides through preliminary screening, as shown in Table 1:
[0030] Table 1 Preliminary screening results of T cell active polypeptides of diphtheria toxin avirulent mutant protein
[0031]
[0032]
[0033] 2) Artificially synthesize the 9 polypeptides described in step 1), mix the obtained 9 polypeptides, emulsify them with an equal volume of Freund's complete adjuvant, and immunize 8 mice, kill the mice on the 10th day after immunization, and take the spleen to prepare a single nuclear lymphocytes.
[0034] 3) Take the mononuclear lymphocytes described in step 2), press 5×10 5 / well was added to a 96-well cell culture plate, and a blank control was set up at the same time. After culturin...
Embodiment 2
[0036] Example 2 Construction of recombinant adenovirus expressing CD137L-PP4 protein
[0037] 1) Artificially synthesized according to the nucleotide sequence encoding the PP4 polypeptide shown in SEQ ID NO.2, the obtained synthetic sequence was inserted into the pDC316 plasmid through the two restriction sites of MluI and HindIII, and the pDC316-CD137L-PP4 recombinant shuttle plasmid was obtained ( figure 2 ).
[0038] 2) Culture HEK293 cells with DEMM medium to grow to monolayer.
[0039] 3) The pDC316-CD137L-PP4 recombinant shuttle plasmid described in step 1) and the adenovirus backbone plasmid pBHGloxΔE1,3Cre were co-transfected with liposome Lipofectamine 2000 into the HEK293 cells described in step 2), and the cytopathic changes were observed under a microscope after transfection In other cases, when the cytopathic changes were completely shed, the primary virus culture medium was harvested.
[0040]4) The primary virus culture solution described in step 1) was coll...
Embodiment 3
[0041] Example 3 Preparation of recombinant adenovirus expressing CD137L-PP4 protein
[0042] 1) Culture HEK293 cells with DEMM medium to grow to monolayer.
[0043] 2) Infect the HEK293 cells described in step 1) with the recombinant adenovirus expressing CD137L-PP4 protein, and harvest the virus culture medium when the cytopathic changes are completely shed.
[0044] 3) Collect the supernatant of the virus culture solution described in step 2) after repeated freezing and thawing three times, and use 300kD membranous cells to carry out tangential ultrafiltration and concentrate 10 times to obtain a concentrated virus solution.
[0045] 4) Purify the concentrated virus solution described in step 3) first with SOURCE30Q anion exchange chromatography, and then with Superdex200pg molecular sieve chromatography to obtain purified virus solution.
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