Tumor adoptosis inducing ligandin and its application in preparing antitumor medicine
An apoptosis-inducing ligand and anti-tumor drug technology, applied in the field of biomedicine, can solve the problem of not being able to obtain the lowest energy, and achieve the effect of small side effects
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experiment example 1
[0045] Experimental example 1, construction of TRAIL-R three-dimensional structure,
[0046] Utilize the HOMOLOGY program package of InsightII of Biosym Company, construct the three-dimensional structure of TRAIL-R by homology modeling method; The homologous protein that chooses is the known three-dimensional crystal structure of human TRAIL receptor (PDB library number: 1d0g, X-ray crystal structure data, the resolution is 0.24nm), using BLAST and FASTA methods to determine the conserved region (SCR) and the loop region (Loop) through sequence comparison, assign coordinates to the corresponding amino acid sequence of TRAIL-R, and obtain TRAIL after adjusting the initial structure - The initial three-dimensional structure of R (as shown in Figure 1 and Figure 3). Select the CVFF force field, and use the DISCOVER-3 program package (such as patent US 6072047) to optimize the molecular mechanics of the modeled protein [5], and go through 18,000 steps, 20,000 steps, and 15,000 ste...
experiment example 2
[0048] Experimental example 2. Obtaining Trail oligonucleotide sequence by mutation
[0049] 1. Proteins expressed by oligonucleotides with the following sequence mutations
[0050] ATG GCT ATG ATG GAG GTC CAG GGT GGT CCG AGC CTG GGT CAG ACC TGC GTGCTG ATC GTG 60 ATC TTC ACA GTG CTC CTG CAG TCT CTC TGT GTG GCT GTA ACTTAG GTG TAC TTT ACC AAC 120 GAG CTG AAG CAG ATG CAG GAC AAG TAC TCC AAAAGT GGC ATT GCT TGT TTC TTA AAA GAA 180 GAT GAC AGT TAT TGG GAC CCG AATGAC GAA GAG AGT ATG AAC AGC CCG TGC TGG CAA GTC 240 AAG TGG CAA CTC CGTCAG CTC GTT CGT AAG ATG ATT TTG CGT ACC TCT GAG GAA ACC ATT 300 TCT ACAGTT CAA GAA AAG CAA CAA AAT ATT TCT CCG CTG GTG CGT GAA CGT GTT CCT CAG360 CGT GTA GCA GCT CAC ATC ACT GGT ACC CGT GGA CGT AGC AAC ACA TTG TCTTCT CCA AAC 420 TCC AAG AAT GAA AAG GCT CTG GGC CGC AAA ATC AAC TCC TGGGAA TCA TCA AGA AGT GGT 480 CAT TCA TTC CTG AGC AAC TTG CAC TTG AGA AATGGT GAA CTG GTC ATC CAT GAA AAA GGT 540 TTT TAC TA...
experiment example 3
[0053] Experimental example 3, the construction of human TRAIL oligonucleotide synthesis and expression plasmid:
[0054] According to the above-mentioned Trail mutation results, the TRAIL oligonucleotide fragments were first synthesized in segments, and then the full-length splicing was performed and sequenced. The synthetic oligonucleotides were subcloned into pGEX-2T plasmid to construct pGEX-2T-TRAIL, and then transformed into recipient bacteria E.coli DH-5α, and the engineering bacteria stably and efficiently expressing TRAIL were screened. Then the PBV220 engineering plasmid was constructed (as shown in Figure 4).
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