Production method and application of Chinese prawn comt recombinant protein
A Chinese prawn and recombinant protein technology is applied to the production method of Chinese prawn COMT recombinant protein and its application field. High enzymatic activity, avoiding the effect of post-treatment
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Embodiment 1
[0031] The expression and purification of COMT recombinase mainly include the following steps:
[0032] ⅰ. Expression: 3 × 10 per shrimp 6 The amount of cfu bacteria, the mixed Vibrio anguillarum ( Vibrio anguillarum ) and Staphylococcus aureus ( Staphylococcus aureus ) live bacteria were injected into the bottom of the telomere of prawns, and after 24 hours of aerated feeding, the total RNA of induced prawn hemocytes was extracted and reverse-transcribed into SMART cDNA as a template, and the synthesis containing Eco R I and not The forward and reverse primers of the I endonuclease site, the primer sequence is:
[0033] COMT ExF: 5'-TACTCA GAATTC ATGTCTTCTCTGAAGAGTTAC-3'
[0034] COMT ExR: 5’-TACTCA GCGGCCGC TCA ATG ATG ATG ATG ATG ATG
[0035] TTTTTTAAAACAGAGAGACAAGC-3'
[0036] Through polymerase chain reaction (PCR), the COMT ORF of 666bp was amplified, and it was cloned into pPIC9K plasmid (Invitrogen), and the recombinant plasmid comt / pPIC9K and pP...
Embodiment 2
[0041] The enzymatic activity of the purified COMT protein was determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The main steps include:
[0042] ⅰ. Preparation of reaction system: Design ①, ②, ③ three groups of 1 mL reaction solution, except 100 mM phosphate buffer (pH7.4), 5 mM MgCl 2 , 200 μM DHBAc, 200 μM SAM (added later), ① group without COMT recombinant protein, as a control; ② group with COMT recombinant protein; ③ group with COMT recombinant protein and internal standard - vanillic acid.
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