Fusion protein, recombinant viral vaccine for treating non-small cell lung cancer and preparation method
A fusion protein and mucin technology, applied in the field of vaccines, can solve the problems of hindering immune response, affecting immunotherapy, and low immunity.
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Embodiment 1
[0060] 1.1 Cloning of human mucin 1 (MUC1) cDNA:
[0061] Collect MUC1-positive human non-small cell lung cancer cells, use total cell RNA extraction reagent Trizol (Invitrogen Company) to prepare cell total RNA, use AMV reverse transcriptase (Promega Company) to synthesize the first strand, use it as a template, and use the following sequence: PCR oligonucleotide primers at the 5' and 3' ends were amplified to obtain a DNA fragment encoding MUCl.
[0062] The 5' end oligonucleotide primer sequence used in the PCR reaction is:
[0063] 5'- GGTACC agccacagccccggt-3' (SEQ ID NO: 10); the horizontal line contains the enzyme cutting site of Kpn I restriction endonuclease;
[0064] The 3' end primer sequence is:
[0065] 5'- GTC GAC ggtgctatggctggc-3' (SEQ ID NO: 11); the horizontal line contains the cutting site of the Sal I restriction endonuclease.
[0066] The reaction parameters were: 95°C for 30 seconds, 62.5°C for 30 seconds, 72°C for 50 seconds, 72°C extension for 10...
Embodiment 2
[0082] 1.1 Cloning of human mucin 1 (MUC1) cDNA:
[0083] Collect MUC1-positive human non-small cell lung cancer cells, use total cell RNA extraction reagent Trizol (Invitrogen Company) to prepare cell total RNA, use AMV reverse transcriptase (Promega Company) to synthesize the first strand, use it as a template, and use the following sequence: PCR oligonucleotide primers at the 5' and 3' ends were amplified to obtain a DNA fragment encoding MUCl.
[0084] The 5' end oligonucleotide primer sequence used in the PCR reaction is:
[0085] 5'- GGTACC agccacagccccggt-3' (SEQ ID NO 10); The horizontal line contains the enzyme cutting site of Kpn I restriction endonuclease;
[0086] The 3' end primer sequence is:
[0087] 5'- GTC GAC ggtgctatggctggc-3' (SEQ ID NO11); the horizontal line contains the enzyme cutting site of Sal I restriction endonuclease.
[0088] The reaction parameters were: 95°C for 30 seconds, 62.5°C for 30 seconds, 72°C for 50 seconds, and after 35 cycles, ...
Embodiment 3
[0111] Use the following pair of primers to perform PCR amplification on the 1146bp insert to obtain a DNA fragment containing a new restriction site. The forward primer has a nucleotide sequence as shown in SEQ ID No.8 in the sequence listing, wherein C AGATCT The part of the horizontal line in the GGagccacagccccggt sequence is the enzyme cutting site of Bg1II; the reverse primer has a nucleotide sequence as shown in SEQ ID No.9 in the sequence table, wherein C AAGCTTG The horizontal line in the sequence of Gtcactcctcgtctttac is the restriction site of Hind III.
[0112] The DNA fragment containing the new restriction site and the pShuttle-CMV shuttle plasmid were double digested with Bg1II and Hind III enzymes, and ligated to obtain a recombinant shuttle plasmid.
[0113] The CEF cells were infected with MVA virus and cultured for 100 min to obtain infected cells.
[0114] After the infected cells were obtained, the recombinant shuttle plasmid was mixed with the infected...
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