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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.

Inactive Publication Date: 2011-11-02
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The object of the present invention is to provide a recombinant melittin protein for treating oviduct diseases of breeders according to the defects of high cost of chemically synthesized antibacterial peptides for treating oviduct diseases of breeders and lack of drugs prepared by biological methods in the prior art

Method used

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  • Recombinant melittin and application thereof
  • Recombinant melittin and application thereof
  • Recombinant melittin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a recombinant melittin and an application thereof, and belongs to the technical field of genetic engineering. An amino acid sequence of the recombinant melittin protein is shown in the formula SEQ ID NO.1. The recombinant melittin protein sequence can be utilized for a preparation of gene medicines utilized for preventing and treating fallopian tubal diseases of breeding hens, in other words, the recombinant melittin protein sequence is connected with a carrier pVAX1 to form a recombinant melittin expression carrier. In addition, a CMV promoter of the carrier pVAX1 is replaced by a chicken ovalbumin gene end 5' regulatory sequence which is an ov sequence, thus the recombinant melittin can be expressed specifically in chicken fallopian tubal tissue. The recombinant melittin and the gene medicines have the advantages of good effects of treating fallopian tubal diseases of breeding hens, safety, no toxicity and broad application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant melittin and its application in preventing and treating oviduct diseases of breeders. Background technique [0002] DNA recombination (Recombinant DNA) technology is the technology of transforming and recombining DNA by artificial means. Including the fine cutting of DNA molecules, the removal of partial sequences, the addition and connection of new sequences, the amplification of DNA molecules, the replication and reproduction of transferred cells, screening, cloning, identification and sequence determination, etc., are the core of genetic engineering technology. Provided technical support for obtaining proteins / peptides (hereinafter referred to as "proteins" and "polypeptides" generically). Many protein drugs, vaccines, and diagnostic reagents have been produced by genetic engineering. [0003] DNA medicine, which is to recombine therapeutically sign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/63C12N15/66A61K38/17A61K48/00A61P31/04
Inventor 谢青梅赵亚华林崇韫毕英佐高向阳马静云陈峰
Owner SOUTH CHINA AGRI UNIV
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