A kind of polypeptide with protease activity and its application
A technology of protease and fusion protein, applied in the direction of application, enzyme, peptidase, etc., can solve the problems of incomplete degradation of protein, protein inactivation, etc.
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Embodiment 1
[0086] 1. Target gene cloning:
[0087] 1. Primer design: use the DrICE gene sequence as a template to design upstream and downstream primers with an NdeI restriction site at the N-terminal and an XhoI restriction site at the C-terminal.
[0088] Upstream primer (SEQ ID NO: 3): GGAATTCCATATGGCTAGAGCCCTGGGCTCCGTGGG Downstream primer (SEQ ID NO: 4): CCGCTCGAGAACCCGTCCGGCTGGTGC
[0089] 2. PCR reaction system: the total volume is 50 μL, of which, 1 μL of Ex Taq DNA polymerase (5U / μL), 5 μL of 10×PCR buffer, 1 μL of upstream and downstream primers (20 μM), 5 μL of dNTP (2.5 mM), template DNA (100ng / μL) 0.5μL, supplemented with ddH 2 0 to 50 μL.
[0090] 3. PCR amplification conditions: 94°C for 5min; 30 cycles of 94°C for 30s, 52°C for 30s, and 72°C for 90s; 72°C for 10min; store at 4°C. The PCR products were subjected to 1% agarose gel electrophoresis (100V, 30min) and the PCR amplification was detected with a gel imaging system.
[0091] 2. Double digestion reaction of PCR p...
Embodiment 2
[0115] 1. Experimental steps:
[0116] 1. Transformation of the carrier
[0117] Using the pET-15b prokaryotic expression vector as a template ( figure 2 ) was transformed, and the N-terminal linker and the DrICE restriction site total 21bp nucleotide sequence (SEQ ID NO: 5CATAGTGATGAAGTTGATGCA) was inserted between the Thrombin restriction site and the NdeI site by PCR ( image 3 ), to obtain the transformed vector pET15D ( Figure 4 ).
[0118] 2. Target gene cloning
[0119] 2.1 Primer design
[0120] Using PYL10 (SEQ ID NO: 6) as a template, the upstream and downstream primers with NdeI restriction site at the N-terminus and XhoI restriction site at the C-terminus are designed as follows:
[0121] Upstream primer (SEQ ID NO: 7): GGAATTCCATATGAATGGCGACGAAACG
[0122] Downstream primer (SEQ ID NO: 8): CCGCTCGAGCTATTCGGCTTGCAGACG
[0123] 2.2 PCR reaction system
[0124] The total volume is 50 μL, including 1 μL of Ex Taq DNA polymerase (5U / μL), 5 μL of 10×PCR buffer...
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