The cDNA sequence and amino acid sequence of the sea earthworm protease with antitumor activity
A technology of anti-tumor activity and protease, which is applied in the field of medical bioengineering, can solve the problem of no new gene sequence of sea earthworm protease, and achieve the effect of inhibiting the proliferation of tumor cells
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Embodiment 1
[0034] Cloning and sequencing of the full-length cDNA of sea earthworm protease.
[0035] Experimental Materials:
[0036] 1. Sea earthworms, collected from the offshore of Yantai City;
[0037] 2. Trizol reagent, 3′RACE kit, 5′RACE kit (Beijing Gibbett), reverse transcription kit (fermentas company), T-AEasy kit, Wizard purified DNA kit, DNase, RNaseH (Promega company), high-fidelity Taq enzyme (Dalian Baobio).
[0038] Experimental method: such as figure 1 shown, including the following steps:
[0039] 1. Design primer sequences: 3R is a reverse transcription primer containing a linker primer sequence; 3R1 is a 3′-end linker primer; 3F1 is a relatively conserved sequence based on annelid phylum earthworms and single-ring echinacea proteases (such as figure 2 :1,2,3,4 are respectively Eisenia fetida DQ836917.1, Eisenia fetida DQ418454.1, Lumbricus rubellus U25647.1, Urechisunicinctus HM623463.1) designed primers. 5R is the oligodT reverse transcription primer....
Embodiment 2
[0079] Construction of recombinant expression vector and expression in Escherichia coli.
[0080] Experimental materials: Endonucleases BamHI and XhoI (Dalian Bao Biology), expression vectors pET-21a, E.coliBL21 are preserved in our laboratory, IPTG (Beijing Dongsheng Taibo), ultrasonic cracker (Shanghai Bilang).
[0081] Experimental method: such as image 3 shown, including the following steps:
[0082] 1. According to the obtained cDNA sequence, design a pair of primers:
[0083] F 5′-TTAT GGATCC ATTGTTTGTGGGTGGGAGGC-3′ R 5′-AATA CTCGAG ACCGATTACGCTATTAACCCAG-3′
[0084] upstream primer F contains BamHI Restriction site, contained in the downstream primer R wxya Restriction sites.
[0085] Using the cDNA of the sea worm protease as a template, pfuDNA polymerase was used to amplify the cDNA fragment without the coding region of the signal peptide, that is, the active peptide.
[0086] 2. The PCR product was connected with the prokaryotic exp...
Embodiment 3
[0090] Isolation and purification of recombinant sea worm protease.
[0091] Experimental materials: nickel column packing, ultrafiltration tube (Beijing Dongsheng Taibo).
[0092] Experimental method: such as image 3 shown, including the following steps:
[0093] 1. Pick a single colony of engineered bacteria, inoculate it in 5ml LB medium containing 100μg / ml ampicillin, and cultivate overnight at 37°C with shaking. Transfer to 500ml of the same medium and continue to culture at 37°C for 4h. Add IPTG (isopropyl-β-D-thiogalactoside) to 1.0mM, continue to culture at 30°C for 4h, and collect the bacteria by centrifugation at 8000×g for 5min.
[0094] 2. Resuspend the bacteria in 25ml Tris-CL buffer (20mM, pH7.4, 10mM imidazole, 0.5MNaCl), sonicate the cells, centrifuge at 12000×g for 30min at 4°C, and take the supernatant.
[0095] 3. Flow the supernatant over Ni 2+ Resin column, washed with 50ml washing solution (20mM Tris-CL, pH7.4, 10mM imidazole, 0.5MNaCl).
[0096] 4...
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