Recombinant melittin and application thereof
A melittin and protein technology, applied in the field of genetic engineering, can solve the problems of lack of drugs and high cost of chemically synthesizing antimicrobial peptides, and achieve the effects of drug safety guarantee and strong bacteriostatic effect.
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Embodiment 1
[0050] Example 1. Preparation of recombinant melittin and its coding gene
[0051] 1. Modified amino acid sequence of melittin
[0052] On the basis of the existing research, the original melittin amino acid sequence was modified to increase the antibacterial activity of melittin while reducing its hemolytic activity.
[0053] The original melittin amino acid sequence is shown in SEQ ID NO:9;
[0054] The modified amino acid sequence is shown as SEQ ID NO: 1, named MLT.
[0055] Change Lys-7 to Arg-7 (enhance the positive charge, and Arg is insensitive to the helix), change Val-8 to Gly-8 (break the helix, increase the hydrophobic moment) and delete the Leu-13 site ( From a thermodynamic point of view, the longer the peptide chain, the longer it takes to fold, and it is not conducive to the combination with the membrane. Leu-13 has nothing to do with the folding of the helix, and deletion of position 13 can significantly reduce hemolysis).
[0056] 2. Obtain the coding ge...
Embodiment 2
[0059] Example 2. Construction of recombinant melittin expression vector pVAX1-MLT
[0060] The MLT gene obtained in embodiment 1 is used Nhe I and Eco R I was digested with double enzymes, and acted at 37°C for 3h. The reaction system is as follows: 10×buffer M 5μL, Nhe I 1 μL, Eco R I 1 μL, MLT gene 25 μL, ddH 2 O to 50 μL, and at the same time pVAX1 plasmid was also treated with endonuclease Nhe I and Eco RI was digested by enzyme digestion, and acted at 37°C for 3h. The reaction system is as follows: 10×buffer M 5μL, Nhe I 1 μL, Eco RI 1 μL, pVAX1 plasmid 20 μL, ddH 2 Make up to 50 μL with O while dephosphorylation with CIAP. All the digested reaction solutions were subjected to 1.5% agarose gel electrophoresis, the bands of the target fragments were excised under ultraviolet light, and the digested target DNA fragments were recovered with a gel recovery kit. Under the action of T4 ligase, the digested pVAX1 vector and the digested MLT gene were li...
Embodiment 3
[0062] Example 3. Construction of recombinant melittin expression vector pOV1.1-MLT
[0063] 1. Amplification of chicken ovalbumin ov sequence
[0064] According to the chicken ovalbumin gene (Chicken Ovalbumine, ov) published on the NCBI database, the full nucleotide sequence length is 9206bp, and the mature mRNA encoding length is 1872bp. (GenBank accession number: J00895) Design upstream and downstream primers, and add restriction enzyme cutting sites at both ends of the fragment, such as SEQ ID NO: 7 and SEQ ID NO: 8, use the total DNA of chicken oviduct as a template, use the above Specific primers amplify the ov sequence. The composition of the PCR reaction system is as follows: 10×buffer 2.5 μL, dNTPs 2 μL, Primers (20 μmol / L) 1 μL each, rTaq DNA Polymerase (2.5U / μL) 0.5 μL, Template 0.5 μL, ddH 2 O to 25 μL. PCR reaction program: 94°C pre-change for 5min, 1 cycle; 94°C for 40s, 55°C for 40s, 72°C for 80s, 30 cycles; 72°C extension for 10min. Using the above produ...
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