cDNA sequence and amino acid sequence of lugworm fibrinolytic enzyme with thrombolysis activity
A kind of sea earthworm and plasmin technology, which is applied in the field of medicine and bioengineering
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Embodiment 1
[0048] Cloning and sequencing of full-length cDNA of sea earthworm fibrinolytic enzyme.
[0049] Experimental Materials:
[0050] 1. Sea earthworms, collected from the offshore of Yantai City;
[0051] 2. Trizol reagent, 3′RACE kit, 5′RACE kit (Beijing Gibbett), reverse transcription kit (fermentas company), T-A Easy kit, Wizard purified DNA kit, DNase, RNase H (Promega company), high-fidelity Taq enzyme (Dalian Baobio).
[0052] Experimental method: such as figure 1 shown, including the following steps:
[0053] 1. Design primer sequences:
[0054] Primer name sequence 3R 5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′ 3F1 5′-GGATCTTGCAATGGTGACAGCGGTG-3′ 3R1 5′-GGCCACGCGTCGACTAGTAC-3′ 5R 5′-TTTTTTTTTTTTTTTTTT-3′ 5F1 5′-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGG-3′ 5R1 5′-CACCCTCTCTGATCCAGTCACGGAAG-3′ 5F2 5′-CCACGCGTCGACTAGTACG-3′ 5R2 5′-GGAGTAGGCAGATGGGTAGGAGGTC-3′
[0055] 3R is a reverse transcription primer containi...
Embodiment 2
[0109] Construction of recombinant expression vector and expression in Escherichia coli.
[0110] 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).
[0111] Experimental method: such as figure 2 shown, including the following steps:
[0112] 1. According to the obtained cDNA sequence, design a pair of primers:
[0113] F 5′-TTAT GGATCC ATTGTTGGTGGCGATGAG-3′ R 5′-AATA CTCGAG GATGTCAGACACCTCTCTGATC-3′
[0114] upstream primer F contains BamHI Restriction site, contained in the downstream primer R wxya Restriction sites.
[0115] Using the cDNA of sea earthworm fibrinolytic enzyme as a template, pfu DNA polymerase was used to amplify the cDNA fragment without the coding region of the signal peptide, that is, the active peptide.
[0116] 2. The PCR product was connected with the pr...
Embodiment 3
[0120] Isolation and purification of recombinant sea worm fibrinolytic enzyme.
[0121] Experimental materials: nickel column packing, ultrafiltration tube (Beijing Dongsheng Taibo).
[0122] Experimental method: such as figure 2 shown, including the following steps:
[0123] 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 1.0 mM, continue to culture at 30°C for 4 hours, and collect the bacteria by centrifugation at 8000×g for 5 minutes.
[0124] 2. Resuspend the bacteria in 25ml Tris-CL buffer (20mM, pH7.4, 10mM imidazole, 0.5M NaCl), sonicate the cells, centrifuge at 12000×g for 30min at 4°C, and take the supernatant.
[0125] 3. Flow the supernatant over Ni 2+ Resin column, washed with 50ml washing solution (20mM Tris-CL, pH7.4, 10mM imidazole...
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