Construction method and application of tumor specific adenovirus vector carrying p21ras single-chain antibody gene
A tumor-specific, single-chain antibody technology, applied in the direction of anti-tumor drugs, applications, antibodies, etc., can solve the problems of low virus delivery efficiency, inability to exert anti-tumor effect, damage, etc., and achieve the effect of reducing damage and clearing effect
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Embodiment 1
[0060] Example 1: Preparation of single-chain antibody gene fragments
[0061] 1. Immunization of Balb / c mice with p21ras protein: Take 5 Balb / c mice aged 6-8 weeks (purchased from the Experimental Animal Center of Chongqing Third Military Medical University), and inject 100 μg of the prokaryotic Express the purified p21ras-K protein (for the preparation method of p21ras-K protein, refer to the paper "Expression, Identification and Purification of Recombinant p21ras Protein and Preparation of Polyclonal Antibody"), add an equal amount of complete Freund's adjuvant for the initial injection, Inject subcutaneously at 5 points. Two weeks later, the second injection was given, with the same dose as the first injection, plus an equal amount of incomplete Freund's adjuvant, and injected subcutaneously at 5 points. Two weeks later, the third injection was given, the dose was the same as the first one, without adjuvant, intraperitoneal injection. The fourth injection was given two w...
Embodiment 2
[0069] Example 2: Establishment and screening and identification of single-chain antibody library
[0070] 1. Construction of recombinant phagemid
[0071] 1.1 Double enzyme digestion of the recombinant pMD-ScFv vector and expression vector: extract the plasmid from the positive pMD-ScFv clone identified by PCR, and follow the instructions of Tiangen Plasmid Extraction Kit for the extraction steps. Recombined pMD-ScFv plasmid and expression vector plasmid pCANTAB-5E (purchased from Pharmacia Company) were digested with Sfi I respectively, and 30 μl of expression vector plasmid / pMD-ScFv vector were added in 200 μl PCR reaction tube respectively; Sfi I enzyme ( 10U / μl) 4μl; 10×Buffer M5μl, ddH 2 O11μl, after the above system is configured, react at 50°C for 4 hours. After gel recovery and purification of the digested product, add 30 μl of the purified product, 2 μl of Not I enzyme (10U / μl), 5 μl of 10×Buffer H, 2 μl of BSA, 2 μl of Trion X-100, ddH into a new 200 μl PCR reacti...
Embodiment 3
[0083] Example 3: Preparation of tumor-specific adenovirus KGHV500
[0084] 1. Construction of tumor-specific adenovirus shuttle plasmid pXC2P
[0085] 1.1 hTERT promoter replaces viral E1A promoter
[0086] 1.1.1 Construction of the E1A gene promoter deletion mutant fragment: using the human adenovirus type 5 shuttle plasmid pXC1 (purchased from Microbix Biosystems Ins) as a template, the E1A promoter deletion fragment was constructed by overlap extension PCR. The specific steps are as follows: Use the E1Af1 forward primer (5'-CGTCTTCAA with the EcoR I restriction site at the end of the primer) GAATTC TCATGTTT-3') and E1Ar1 reverse primer (5'-CCGCTCGAGCGGCGAC GTC GAC GCGTCACTACACGTCAGCTGACTATAATAATA-3') amplifies the partial fragment 894bp upstream of the E1A promoter. The E1Af2 forward primer (5'-ACGCGTCGACGTCGGCCG with the Xho I restriction site at the end of the primer CTCGAG CGG GACTGAAAATGAGACATATTATCTGC-3') and E1Ar2 reverse primer (5'-TGCATTC TCTAGA CACAGGTGAT-3...
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